652 

;5 
•py 1 



SOIL INOCULATION WITH 
AZOTOBACTER 



BY 
PAUL EMERSON 



DISSERTATION SUBMITTED TO THE GRADUATE FACULTY OF 
THE IOWA STATE COLLEGE OF AGRICULTURE AND ME- 
CHANIC ARTS IN PARTIAL FULFILLMENT OF 
THE REQUIREMENTS FOR THE DEGREE 
OF DOCTOR OF PHILOSOPHY 

NO. 3 



REPRINTED FROM RESEARCH BULLETIN No. 45 

IOWA AGRICULTURAL EXPERIMENT STATION 

1918 



Digitized by the Internet Archive 
in 2010 with funding from 
The Library of Congress 



http://www.archive.org/details/soilinoculationwOOemer 



February, 1918 Research Bulletin No. 45 



Soil Inoculation with Azotobacter 



<^ LSZ 



By PAUL EMERSON 



AGRICULTURAL EXPERIMENT STATION 

IOWA STATE COLLEGE OF AGRICULTURE 

AND MECHANIC ARTS 



AGRONOMY SECTION 

Soil Bacteriology 



AMESs IOWA 



IOWA AGRICULTURAL EXPERINENT STATION 
OFFICERS AND STAFF 

Raymond A. Pearson. M. S. A., LL. D.. President 

C. P. Curtiss. M S. A., D. S.. Director 

W. H. Stevenson. A. B.. B. S. A., Vice-Director 
AGRICULTURAL, ENGINEERING 
C. K. Shedd, B. S. A., B. S. in A. E., W. A. Foster, B. S. in Ed., B. Arch., 
Acting Chief Assistant 

AGRONOMY 
W. H. Stevenson. A. B., B. S. A., Cliief George E. Corson, B. S., M. S'., As- 
H. D. Hiiglies. B. S., M. S. A., Cliief sistant in Soil Survey 

in Farm Crops H. W. "Warner, B. S.. M. S., Soil Sur- 

P. E. Brown, B. S.. A. M.. Ph. D., Chief veyor (ab.=-ent on leave) 

in Soil Chemistry and Bacteriology -^ l, Rhodes, B. S., Soil Survey (ab- 
L. C. Burnett. M. S. A., M. S., Chief ^ent on leave) 

in Cereal Breeding .. , ^, . „ M. E. Olson, B. S., M. S'., Field Ex- 

L. W. Forman. B. S. A., M. S., Chief periments 

in Field Experiments . , ^ EI. Angell. Soil Surveyor 

John Buchanan, B. S. A Supermtend- j p gjg-g. g g pie\& Exnerimenta 
ent of Co-operative Experiment.^ q p j^^^^^,-, g g., m. S., Assistant 
R. S. Potter A, B.. M. b.. Pn. u.. . ^^ rmno 

Assista-t Ciiipf in Soil Chemistrv ^ ' Hnn=-on B q Eipli Exneri- 

R g S'lvdpr. B. S., Assistant in Soil H. P. Hanf:on. B. ^-^ -l^ield iixpen 

pviop^isfv ments (absent on leave) 

H. "^'■. .Tobn^nn B S.. M. S . Assist- 
ant in Soils (absent on le^-'^o'i 

ANIMAL HUSBANDRY 
W H Pew B S A Chief L. S. Gillette. B. S.. M. S., Assistant 

J. M. Evvard, M, S., Assistant Chief Chief in Dairy Husbandry _ 

in Animal Husbandry and Chief in A. C. McCandlisb, M. S. A., Assistant 
Swine Production in Dairv Husbandry 

R. Dunn. B. S., Assistant in Animal Rodney Miller. B. S. A., Assistant in 

Husbandry Poultry Husbandry 

H. A. Bittenbender, B. S. A., Chief 
in Poultry Husbandry 

BACTERIOLOGY 
R. E. Buchanan, B. S., iPh. D., Chief : Associate in Dairy and Soil Bacteriology 

BOTANY 
L. H. Pammel, B. Agr., M. S., Ph. D., I. E. Melhus, Ph. D., Chief in Plant 
Chief Patbolo.gy 

Charlotte M. Kin?, Assistant Chief in Botany 

CHEMISTRY 

A. W. Dox, B. S.. A. M., Ph. D., Chief S. B. Kuzirian, A. B., A. M., Ph. D., 

(absent on leave) Assistant 

W. G. Gaessler, B. S., Acting Chief G. W. Roark. Jr., B. S., Assistant 

A. R. Lamb. B. S., M. S'.. Assistant Lester Yoder, B. S., M. S'., Assistant 

DAIRYING 
M. Mortensen. B. S. A., Chief D. E. Bailey. B. S., Assistant Chief 

B. W. Hammer, B. S. A., Chief in in Dairying 
Dairv Bacteriology 

ENTOMOLOGY 
R. L. Webster, A. B., Acting Chief Wallace Park, B. S,, Assistant in Ag- 

riculture 
FARM MANAGEMENT 
H B Munger, B. S., Chief O. G. Lloyd. B. S., M. S'., Assist. Chief 

HORTICULTURE AND FORESTRY 
S. A. Beach. B. S.. M. S., Chief J. B. Kendrick, B. A., Research As- 

T. J. Maney, B. S., Chief in sistant in Pomology 

Pomology ' A. T. Erwin, M. S., Chief in Truck 

Harvey L. Lantz. B. S., Assistant in Crops 

Fruit Breeding Rudolph A. Rudnick, B. S., Assistant 

W. E. Whitehouse, B. S., Assistant in Truck Crops 

in Pomology G. B. McDonald, B. S. F., M. F., Chief 

Andrew Edward Murneek, B. A., Re- in Forestry 

search Fellow in Pomology Frank H. CuUey. B. S. A., M. L. A., 

Chief in Landscape Architecture 

RURAL SOCIOLOGY 

G. H. Von Tungeln. Ph. B., M. .\., Chief 

VETERINARY MEDICINE 

C. H. Stange, D. V. M., Chief 

GENERAL OFFICERS 

F. W. Beckman, Ph. B., Bulletin Editor F. B. Colburn, Photographer 

Gretta Smith. A. B., Assistant to Bulletin Editor 

C, E. Brashear,_B'._S. A., Assistant to Director 

""" JSaRYc QF CONGRESS j 

. .RECEIVED 






Soil Inoculation With Azotobacter* 

BY PAUL EMERSON. 

Following' tlie discovery of the nitrogen fixing' powers of the 
symbiotic bacteria in the soil, early investigators found that the 
power of utilizing the free atmospheric nitrogen was not confined 
to the symbiotic bacteria alone. They noted increases in soils 
which had borne no legumes and they found that fallow soils 
in particular increased appreciably in nitrogen content. These 
facts stimulated researches which led to the discovery of many 
forms of bacteria which are able, when growing alone, to fix 
nitrogen from the air. The chief of these is now known as the 
azotobacter group. 

It seems likely that the azotobacter will prove more effective in 
fixing nitrogen than the symbiotic bacteria, although the general 
requirements of the two classes of organisms are very similar. 
The azotobacter are active in practically all soils regardless of 
the kind of crop grown when conditions for their growth are 
satisfactory. These conditions are probably much the same as 
for the symbiotic bacteria except that these latter organisms 
require the presence of a specific legume for fixing the greatest 
amount of nitrogen. Azotobacter require a certain amount of 
carbonaceous material in the soils and are usually stimulated by . 
a small amount of nitrogen, but the exact optimum conditions 
for their growth are as yet unknown. These organisms are active 
in causing nitrogen increases in many soils, but the feasibility 
of introducing them into the soil or of attempting to increase 
their nitrogen-fixing powers by artificial means, and the effect 
of the presence of growing plants on their efficiency are ques- 
tions as yet unanswered, although Lipman has indicated that 
under proper conditions successful inoculation may be accom- 
plished in soils and Bottomley has successfully grown pure cul- 
tures of these organisms in the presence of growing plants with 
favorable results. 

HISTOBIGAL 

Beijerinck (2) isolated and described the first azotobacter (in 
1901) . He found two species, one of which he named Azotohacter 
chroococcum and the other Azotohacter agilis. The former was 
isolated from the soil and the latter from a sample of water 
taken from one of the canals in the city of Delft. Two years 
later Lipman (36) added a third species, A. vinelandii, to the 
list and the following j^ear isolated and described two more, giv- 
ing them the names of A. deijerinckii and A. woodstownii. Of 
the five organisms of this species, A. cliroococcum., A. deijerincMi 

♦Thesis submitted in partial fulfillment of the requirements for the Degree 
Df Doctor of Philosophy at the Iowa State College. 

'■h ■'>«<•■ ...' 



28 

and A. vinelandii are considered the most important in soil in- 
oculation studies. 

The frequency with which investigators in all parts of Europe 
and America have isolated azotobacter from various soils, indi- 
cates that they are widely distributed. Christensen (10) found 
that they were present throughout northwestern Europe, the 
activity of the organism apparently depending on the basicity 
of the soil. This view was later supported by the works of 
Fisher (14), Lohnis and Pillai (45) and others. Ashby (1) 
studied the soils of Mombasa, East Africa, Cairo, Egypt and 
Rothamsted, England and found azotobacter forms present in 
most eases. Lipman and Burgess (42) working with forty-six 
samples of soil from variotis parts of the world, found that over 
one-third of them contained azotobacter, the predominant form 
being A. chroococcum. Many of the soils examined were museum 
specimens and had been kept in tightly stoppered bottles for 
long periods of time. 

DESCRIPTION OF AZOTOBACTER. 
Bei.jerinck characterizes the azotobacter r-s stout bacteria, 4-6 
microns or less in length, sometimes longer, occurring as large 
diplococci or short rods in young cultures, the hyaline cells often 
containing a vacuole and the entire organism enclosed in a 
mucilagenous wall of varying thiclaiess. They have a single 
polar flagellum or bundles of 4-10 polar flagella of about the 
same length as the organism itself. Beijerinck found no spores. 
Vagler (65) writes that the older colonies produce involution 
forms similar to those of yeasts while Heinz (22) and Fisher 
(15) showed that the organisms can resist drying for six to nine 
months. Later investigations by Mulvania (50) and Lohnis and 
Smith (47) demonstrated that the organism produces spores and 
completes a very complicated life cycle. Descriptions of azoto- 
bacter and detailed cultural characteristics of the organism were 
given by Lipman (35), Bei.jerinck (2), Prazmowski (54), "Warm- 
bold (70), Bonazzi (6), Lohnis and Westerman (48), Lohnis and 
Hanzawa (44), Jones (27) and others. 

ACTIVITIES OF AZOTOBACTER. 
Beijerinck first claimed that the isolated pure cultures of 
azotobacter were able to fix the atmospheric nitrogen in appre- 
ciable amounts; later, however, when working with Van Delden 
(4), he retracted this statement, claiming that pure cultures did 
not have this ability and that only in the presence of very small 
celled organisms called radiobacter could the free nitrogen of 
the air be fixed in the soil. Grcrlach and Vogel (18), Heinz 
(23), Lipman (37) and Freudenreich (17) proved conclusively 
that the earlier conclusions of Beijerinck were correct and that 



29 

the organism may fix considerable amounts of nitrogen in pure 
cultures. Lipman accounts for the fact that Beijerinck did 
not get a fixation of nitrogen in pure cultures by showing that 
the organism will not fix nitrogen unless the reaction of the 
medium is made neutral or slightly alkaline. When Beijerinck 
later accepted this suggestion he found that his pure cultures 
were able to fix atmospheric nitrogen. 

STUDIES OF AZOTOBACTER. 

Ver}' few investigators have attempted tO' inoculate soils with 
azotobacter or other non-symbiotic nitrogen fixing bacteria 
under conditions approximating those in the field. The influence 
of various kinds of sugars, cellulose, inorganic salts, and various 
organic compounds on the nitrogen-fixing power of the organ- 
isms have been studied extensively. Gerlach and Vogel (19), 
Pringsheim (55), Krainsky (33), Koch (30), Hoffman and Ham- 
mer (25) and Stranak (61) have found that various sugars and 
cellulose materially increase their nitrogen fixing powers while 
Fisher (16), Christensen (10), Lohnis and Pillai (46), Wilfarth 
and Wimmer (59) Kaserer (28), Rosing (59), Vogel (66), 
Greaves and Anderson (20) and Pringsheim (56) have shown 
that small amounts of lime, very small amounts of nitrogen, 
various inorganic salts and even a very small amount of arsenic 
will stimulate the nitrogen fixing power of the organisms in the 
presence of certain carbon compounds. Stoklasa (60) studied 
the products of the activities of the azotobacter organisms, con- 
fining his researches largely to the amounts and kinds of gases 
produced under different circumstances, under the influence of 
various substances supposed to be energy sources, and under 
varying temperature conditions. His results have been more or 
less confirmed by the works of Thiele (64), Hoffman (24), Keller- 
man and Smith (29) and Ehrenberg (13). 

The activity of the azotobacter in soils in general, and partic- 
ularly under laboratory conditions, was fully shown by the works 
of Lipman (39), Voorhees and Lipman (68), Lohnis (43), Kuhn 
(34), Freuclenreich (17), Dvorak (12), Remy (57), Remy and 
Rosing (58), Jacobitz (26), Stranak (62), Headden (21), Peter- 
son and Mohr (52), Koch and Seydel (31), Omeliansky and 
Ssewerowa (51), Warmbold (71) and others who demonstrated 
that under various conditions and in almost every type of known 
soil these organisms are able to fix appreciable amounts of the 
free atmospheric nitrogen. Only a few of these investigators, 
however, have made any attempt to secure an active flora of 
these organisms in the soil. Vogel (67) inoculated pure cultures 
of azotobacter into soils that had been treated with grape sugar, 
in some cases adding comparatively large amounts of nitrate of 
soda. In pot experiments with oats and mustard, increases were 



30 

noted for the inoculated series, altho the pots receiving nitrate 
of soda gave the greatest yields. When the experiment was re- 
peated in the field the inoculated plots gave smaller yields than 
the uninoculated, and the inoculation appeared to have an in- 
jurious effect upon the crop. 

A short time later Lipman and Brown (41) tried inoculation 
experiments with A. vinelandii and A. beijerinekn. They sunk 
four foot cylinders open at both ends into- the soil, filled the 
cylinders with soil and inoculated the soil with the organisms. 
The first summer the soils were left bare and then a rotation of 
crops was followed and oats, corn and rye grown in succession. 
While considerable variation Avas found in the nitrogen content 
of the crops and in the dry weight, the general conclusion reached 
was that the activities of the organisms did not increase the 
nitrogen content of the soil. The results do not preclude the pos- 
sibility that inoculation with the organisms in question may be 
made of practical value, provided proper conditions for the best 
growtli of the organisms are secured. Bottomley (7) and Bot- 
tomley and Hall (9) experimented with oats, barley and some 
root crops, and arrived at the same conclusions as did Lipman 
and Brown. Stranak (63) also inoculated soils with azotobacter 
and found a pronounced increase in the growth of potatoes, 
grain and beets. 

Altho the experiments dealing with the inoculation of soils 
with azotobacter have been inconclusive, it is believed that under 
proper conditions such inoculation may be extremely profitable. 

EXPERIMENTAL 

The wdde distribution of non-symbiotic nitrogen fixing bacteria 
in many types of soils is practically parallel with the distribution 
of the symbiotic organisms, and since it is practical and profitable 
to inoculate soil with the latter, even tho the particular organism 
may be present, the following cjuestions quite naturally arise: 

1. If the azotobacter are not present in the soil, can inocula- 
tion be profitably accomplished? 

2. What soil conditions are necessary for the greatest fixa- 
tion of nitrogen by these organisms ? 

These questions have an important bearing on the problem of 
the maintenance of permanent fertility in soils from the nitrogen 
standpoint and may govern the choice of the proper method of 
farming. Some commercial concerns have placed cultures on the 
market, claiming that they contain sufficient numbers of the non- 
symbiotic nitrogen fixing bacteria to enable the farmer to solve 
his nitrogen problem without growing legumes. However, results 
of experiments showing that such cultures are capable of inoculat- 
ing the soil were not found in the present investigation. 



31 

INCREASING THE NITROGEN FIXING POWER OF PURE 

CULTURES. 

Very little work has been done along the line of breeding pure 
cultures of bacteria to an increased efficiency in their specific 
actions, in fact, practically all the experiments have been carried 
out with the idea of finding a method whereby the organism 
could be kept alive for long periods without periodic transfers. 
The earliest investigation along this line was that of Czaplewski 
(11) who limited the amount of air in the tube by saturating the 
plug with paraffine. Later Lunt (49) found that certain cultures 
of water bacteria may be kept alive much longer in sterile water 
than in ordinary culture media. In some cases he kept certain 
organisms alive for two years by this method. BoUey (5) secured 
good growths of B. amylovorus and Bad. dianthi in agar and 
in bouillon by making transfers from cultures that had been 
hermetically sealed for nine years. It is not stated whether or 
not the organisms were tested for their pathogenicity and hence 
their virulence is left in doubt. This work supports that of 
Czaplewski in showing that cultures can be kept alive for long 
periods of time if the transpiration is reduced to a minimum. 
Some commercial concerns claim that they are able to increase 
the efficiency of their particular cultures of legume bacteria by 
alternate inoculations first on agar, then into sterile greenhouse 
soil, growing the specific legume to which the organism in ques- 
tion is adapted, and re-isolating the crsanism from the nodules 
produced on the roots of the legume. If this is possible for the 
symbiotic bacteria then it seems probable in the case of the non- 
symbiotic organisms. The following cpiestions naturally suggest 
themselves : 

1. Can the nitrogen fixing power of azotobacter be increased 
by periodic transfers on nitrogen free media? 

2. Can the nitrogen fixing powers of azotobacter be increased 
by growing the organism in the presence of growing plants ? 

In outlining work to answer the above questions it was realized 
that a large number of bacteria should be used. A number of 
large celled nitrogen fixing organisms that had all the staining 
reactions of the azotobacter type and closely resembled it in size 
and shape, were isolated in pure cultures from soil secured from 
the humus plots at the Iowa station and were designated with 
laboratory numbers. At the same time pure cultures were se- 
cured and their activities determined along with these of the 
unnamed cultures. The pure cultures were kindly furnished by 
Dr. J. G. Lipman of the New Jersey Agricultural Experiment 
Station and also by the American Museum of Natural History of 
New York, 



32 

MEDIA USED. 

The nitrogen free medium used thruout the experimental 
work was a modification of that proposed and used by Lipman 
(35), and its composition was as follows: 

Distilled water 1,000 cc 

Di-potassium phosphate 0.2 grams 

Magnesium sulphate 0.2 grams 

Calcium chloride 0.02 grams 

Dextrose 10.0 grams 

10% Ferric chloride solution 2 drops 

The solution was brought to boiling and made neutral to 
phenolphthalein by the addition of N/10' NaOH. If a solid 
medium was desired 1 % powdered agar w^as added. Sterilization 
was accomplished by placing in the autoclave at ten pounds for 
20 minutes. 

Inoculation was secured by scraping off a two days' growth 
from the agar slants with a sterile needle and transferring it to 
flasks containing 50 cc. of the above solution. In onler to de- 
termine w^hether the nitrogen fixing power of the organisms was 
stimulated by the addition of nitrogen, the above solution with 
the addition of 1 mg. of nitrogen as sodium nitrate was used. 

PRELIMINARY STUDIES. 

All of the organisms of the azotobaeter type including both 
the pure cultures and the unnamed cultures, were inoculated 
into 50 c. c. of both of the above solutions and tested for their 
nitrogen fixing powers. The inoculated solutions were incubated 
for tliree weeks at room temperature (22-25° C) and then 
Kjeldahlized. The amount of nitrogen fixed by each organism 
in the different solutions is shown in tal>le I. The same cultures 
were transferred 12 times at three to four day intervals on nitro- 
gen free media and their nitrogen fixing power tested in solu- 
tioiis with and without nitrogen. The results appear in table II. 

The laboratory organisms used in table I had been freshly 
isolated and purified from the soil, the named cultures had been 
kept on agar slants for varying periods of time. During the 
time that the inoculated culture solutions were incubating the 
transfers were being made in preparation for the inoculations 
for table II. 

Comparing the two tables we find that a ma.jority of the or- 
ganisms decreased in their ability to fix atmospheric nitrogen, 
altho a few showed a slight increase or at least retained their ef- 
ficiency. From these the following eight were selected for fur- 
ther study: No. 4, No. 22, No. 26, No 27, A. vinelandn, A. chroo- 
cocciim., A. heijerincMi and A. cliroococcum (HCM) . These eight 
organisms were studied under both laboratory and greenhouse 
conditions, 



33 



TABLE I^NITEIOGIEN-TTKATTON OBT 

PUBE ClUXiTUKES IN SQiLUTION 

WITH AJNID WITHODT 

MTKiOGEN. 





N. Fixed in Mgs. 


Organism 


c " ?, 

■sjl 


Solution 

with 

Nitrogen 


Lab. No. 1 . - 


2.24 
2.38 
0.28 
0.98 

i.9e 

3.22! 
0.42 
0.42 
0.42 
lost 

1.12 

2.80 
7.141 
0.2S 
1.12 
0.56 
0.50 
0.42 
0.70 
0.70 
0.2S 
0.56 
a. 12 
lost 
4.20 
0.84 
0.70 
0.84 
1.96 


V.U 


Lab. No. 2- 


1.54! 


Lab. No. 3 


1.99 


Lab. No. 6 

Lab. No. 7 -__ 


1.82 

1.68 


Lab. No. & 


3.10 


Lab. No. 10 


0.84 


Lab. No. 11 


0.70 


Lab. No. 12 - 


1.40 


Lab. No. 14 

Lab. No. 15 


lost 
1.13 


Lab. No. le - - 


lost 


Lab. No. IS 

Lab. No. 19 - __ 


U.43 

1.54 


Lab. No. 20- -. 


1.90 


Lab. No. 21 __ ... _ 


2.10 


Lab No. 22 


2.53 


Lab. No. 23- - . -- 


2.52 


Lab. No'. 24- 


1.82 


Lab. No. 25- - 


1.82 


Lab No. 26- 


2.10 


Lab. No. 27 

A. vinelandii 


5.60 
2.66 


A. chroo'coecum (IHCIM) 

A. chroocoecum 

A. chi'Oococeum (Oolo). 
A. beijerinekii 


3.08 
1.54 
2.52 
1.68 


A. bsijerinekii No. 5 


2.38 



TAiBiLE n—NITBOOEN- FIXATION BT 

FORE! COLTURES IN SOLUTION 

WITH AND WITHOUT 

NITRIOGEN. 

After each org-anism has been transfer- 
red twelve times on nitrogen- free 
miedia at three to four day intervals. 





N. Fixed in Mgs, 


Organism 


3 M 


.2 S, 


Lab. No. 1 -- 


0.84 
0.14 
0.14 

1.82 
0.00 
0.28 
O.OO 
0.98 
0.00 
0.00 
0.42 
0.00 
O.OO 
0.00 
0.00 
0.98 
0.42 
3.'53 
2.10 
0.00 
0.00 
1.12 
1.40 
0.00 
1.12 
2.52 
O.OO 
0.42 
1.12 


1.12 


Lab No'. 2 


O.OO 


Lab. No. S 


0.99 


Lab. No. 4 


0.98 


Lab. No. 6 _ 


0.84 


Lab No. 7 - 


0.28 


Lab No. 9- 


1.26 


Lab. No. 10 


0.00 


Lab No. 11 - - 


1.6S 




0.28 


Lab. No. 14- 


1.121 


Lab No. 15 


1.40 


Lab No. 16- -— 


0.14 


Lab No 18 


0.42 


Lab. Noi. 19 --- 


0.42 


Lab No 20 


1.54 


Lab. No. 21 


0.84 


Lab. No. 22 

Lab No. 23 


1.12 
1.54) 


Lab No 24 


1.63 


Lab No. 25 


0.2S 


Lab No. 26. - - 


1.82 


Lab. No. 27- 


2.52 
0.00 


A. chroocoecum 

A. chroocoecum (HICM) 
A. chroocoecum (Colo). 


1.82 
1.12 
1.26 
2.92 


A. beijerinekii No. 0.— 


0.00 



LABORATORT STUDIES. 



The laboratory studies were arranged in a series of three ex- 
periments as follows : 

1. To determine the effect of transfers made every other day 
on the nitrogen fixing- power of the organisms. 

2. To determine the effect of transfers made once each week 
in sand cultures variously modified. 

3. To determine the effect of growing four of the organisms 
on both agar and sand in large flasks with and without the 
presence of growing plants. 



34 

Series 1. To Determine the Effect of Transfers Made Every 

Other Day on the Nitrogen Fixing Power of 

the Orga!msms. 

Using the eight selected organisms transfers were made every 
other day on the nitrogen free medium for a period of three 
weeks. It was feared that such rapid transferring for so long 
a period on a medium practically free from nitrogen would re- 
duce the vitality of the organisms, accordingly each fifth trans- 
fer was made on a modification of the medium consisting in the 
addition of one milligram of nitrogen as sodium nitrate to each 
liter of the regular dextrose agar. At the end of the transfer 
period the organisms were inoculated into the nitrogen free and 
nitrogen containing solutions incubated for the same periods of 
time and the amount of nitrogen fixed determined by Kjeldah- 
lizing. The results of the determinations are shown in table III. 

TABIE nr— TIHiE EPFEiOT OF TIHuANiSIEEiHlS iMADE: EVElE;T OTHER DAT FOB FOUIB 
WEEOBCS ON THE' NTTRlOGEiN MXINIG POWEIB OF THE OCROAJSfTSMS . 



Organism 



Nitrogen Fixed in Mgs. 



Solution without 
Nitrogen 



Solution with 
Nitrogen 



Lab. No. 4 

Lab. No. 25 

Lab. No. 26 

Lab. No. 27 

A. vinelandii 

A. chrooeoecum 

A. ehrooeoccuni CHOM). 
A. beijerinckii 



(a) 


(b) 


(Av.) 


(a) 


(b) 


0.14 


0.42 


0.28 


0.70 


0.42 


0.00 


O.OO 


0.00 


0.14 


0.56 


0.1+ 


0.14 


0.14 


0.28 


0.98 


0.2S 


3.. 50' 


1.98 


0.70 


0.98 


0.28 


0.42; 


0.35 


0.98 


0.98 


r^r. 


0.2S 


0.42 


0.98 


1.12 


2.66 


lost 


2.66 


1.40 


1.40 


0.S4. 


2.66 


1.79 


0.28 


0.28 



(Av) 
0.56 
0.35 
0.63 
0.84 
0.98 
l.Oo 
1.40 
0.28 



That these transfers should have been made at longer intervals 
is evidenced by the fact that tables I and II showed that 12 of 
the cultures had increased in efficiency after they had been 
transferred every three days for 36 days. However, during the 
latter work the organisms did not show any indications of a loss 
of vitality and the growth at all times was vigorous and rapid. 
Tabic III shows a decrease in the nitrogen fixing powers of all 
the organisms except in the case of A. diroococcum (II CM) 
which appears to have retained its efficiency thruout the ex- 
periment. 

Series 2. To Determine the Effect on the Nitrogen Fixing Power 
of Transfers Made Each Week in Sand Cultures. 

In the following experiment sand was used instead of agar as 
the basis for the medium. Ground oats straw, ground red clover 
hay and either the regular dextrose solution, or the dextrosf; 
solution containing nitrogen were added. The tests were carried 
out in tubes arranged as follows : 



35 



6.25 gr. sand+2.5 cc N. free dextrose solution. 

6.25 gr. sand-i-2.5 cc dextrose solution containing 0.2 gr. NaNOs per 

liter. 
6.25 gr. sand + 3.5 cc N. free dextrose solution + 0.1 gr. clover nay. 
6.25 gr. sand-|-3.5 cc N. free dextrose solution+0.1 gr. oats straw. 

The organisms were transferred directly from the slants into 
the tnbes and there allowed to incubate at room temperature for 
seven days. A small portion of the sand was then transferred 
to a fresh tube of the same medium as the original. As this par- 
ticular experiment did not directly follow the others the efficiency 
of the organisms was tested before they were inoculated into the 
sand. Table IV shows the amount of nitrogen fixed by the pure 

TAjB^LlE' IV-^TIBTE NITEiOOEN MXINO POWEIK OlF THE- PORIE CICPLiTUEElS 
IMMEIDIAT'ElLY BiEIPIOBIE: the SIANID' CIU'LITHJIRE' EXPEIRIIlMElN'TlS. 



Organi; 



[Solution witnout 
Nitrogen 



Solution with 
Nitrogen 



Lab. No. 4 

Lab. No. 22 

Lab. Nc 26 

Lab. No. 27 

A. vinelandii 

A. ehroococcum 

A. chroococcum (HJQM)- 
A. beijerinckii 



(a) 

0.14' 
0.00 
O.OO 
0.00 
O.OO 
O.OO 
0.00 
0.98 



(b) 
0.70 
0.00 
0.14) 
0.00 
0.00 
0.14 
O.OO 
0.84 



(Av.) 
0.42 
O.OO 
0.07 
O.OO 
0.00 
0.07 
0.00 
0.91 



(a) 


(b) 


0.28 


1.54 


3.36 


3.0s 


2.914 


2.66 


2.38 


2.52 


1.40 


1.40 


2.80 


2.66 


2.52 


2.38 


2.94 


lost 



(Av. 
0.91 
3.22 
2.80 
2.45 
1.40 
2.73 
2.49 
2.94 



cultures at the beginning of this series of incubation, and the 
same methods as in the previous experiments. 

At the end of the fourth transfer period, i. e., four weeks 
after the start of the experimental series, the organisms were 
inoculated into dextrose solution and their nitrogen fixing powers 
determined. After four more weeks of transferring or in all 
eight weeks the final inoculation into dextrose solution was made. 
The influence of the oats and clover in the presence of sand on 
the nitrogen fixing power of the organisms used is shown in tables 
V and VI, by the fact that both the large celled organisms of the 

TAtBLE v.— NTTIBOiGEN FIXED BY THE PUBE COLTIPREIS APTE-B FOUR 
TBAiNSFElBS IN ISLAND' AT PElRilODS! 'OF' SEVEN D'AYS EACH. 





Nitrogen Fixed in Mgs. 


Organism 


dex. sol. 


dex. sol. 

+ N 


dex. S0I.+ 
oats straw 


dex. S0I.+ 
clover hay 


Lab No. 4 


0.28 
0.07 
0.77 
0.42 
0.14 
0.21 
O.07 
0.14 


0.35 
0.2s 
0.07 
0.351 
0.21 
0.21 
0.14 
0.28 


0.28 
0.00 
0.14 
1.27 
0.42: 
0.07 
1.19 
0.35 


0.98 


Lab. No. '» 


0.35 


Lab. No. 26 

Lab. No. 27 


0.00 
0.42 
0.28 


A. ehroococcum 

A. chroococcum) (HCIM) 

A. beijerinckii 


0.21' 
0.42 
0.28 



36 



"HAiBLE' VI— NITRlOOEilSr iPIXED EiT THE FUBIE' OULTUElElS AFT'EIB EIGHT 

THAaSHSE'EIRlS AT PiElRODOIDlSi OP SEiyEN DAYS- EACH. 



Organism 



Lab. No. 4 

Lab. No. 23 

Lab. No. 20 

Lab. No. 27 

A. vinelandii 

A. chrooeoccum 

A. chrooeoccum! (HCiM) 
A. beijerinckii 



Nitrogen Fixed in Mgs. 



dex. sol. 



0.30 

0.20 
0.70 
0.20 
0.20 
0.30 
0.10 
0.00 



dex. sol. 


dex. sol.+ 


+ N 


oats straw 


0.20 


0.30 


0.20 


0.20 


0.40 


o.oo 


0.10 


0.40 


0.40 


0.40 


o.ao 


0.20 


0.40 


0.40 


0.00 


0.20 



dex. S0I.4- 
clover hay 



0.40 
2.00 
1.00 
0.50 
0.50 
lost 
1.40 
O.b'O 



azotobaeter type and the azotobaeter themselves, made gains in 
their nitrogen fixing powers. There was no distinct gain due 
to any one kind of carbonaceous material. Of the six organisms 
showing gains A. cJiroococcum made the most notable, especially 
in the presence of the oats straw. The nitrogen fixing power of 
No. 4 appears to be rather constant thruout the series, with no 
appreciable gain or loss. A. heijerinckii showed a decided loss in 
its power to fix nitrogen in each of the four media, but gave a 
slight indication that in the presence of the clover hay it might 
be slowly regaining its power. 

Series 3. To Determine the Effect of Growing the Organisms on 

Both Agar and Sand With and WitJiont the 

Presence of Growing Plants. 

The main points considered in this experiment were: An 
increase in the surface area over which the organism could 
grow; an increase in the time between transfers and the grow- 
ing of the organisms in the presence of an undetermined species 
of alga3 and with growing oats and red clover plants. Two liter 
Erlenmeyer flasks were used and arranged in the following man- 
ner conforming to the outlines of the experiment: 

Flask No. 1. 1000 cc N. free dex. agar+1 gr. CaCOj planted to oats. 

Flask No. 2. 1000 cc N. free dex. agar-fl gr. CaCO 3 planted to 
red clover. 

Flask No. 3. 1000 cc N. free dex. agar-f 1 gr. CaCOa planted with 
an undetermined species of algse. 

Flask No. 4. 1000 gr. pure quartz sand + 180 cc N. free dex. solution 
neutralized with CaCO:!, planted with oats. 

Flask No. 5. 1000 gr. pure quartz sand + 180 cc N. free dex. solu- 
tion neutralized with CaCOs, planted with red clover. 

Flask No. 6. 1000 gr. pure| quartz sand+180 cc solution without 
dex. neutralized with CaCOs, planted with an undetermined species 
of algae. 

Check flasks of sand and dextrose agar. 



37 

The flasks of agar were sterilized in the autoclave at ten pounds 
for 30 minutes, but the flasks of sand were sterilized at 15 pounds 
pressure for four hours once a day for three consecutive days. 
Bacteriological tests on the sand at the end of that time showed 
it to be sterile. 

The culture of the algae used was so closely associated with a 
bacterial growth that a separation would have required a long 
time. For that reason it was not purified but was grown in sterile 
distilled water, for about three months before inoculation. The 
inoculation of the algae was made in the flasks about two weeks 
before the inoculation vrith the azotobacter cultures in order that 
the algae might make a sufficient gTowth to supply Vie bacterial 
cultures with the proper amount of carbonaceous material. To 
prevent contamination by the oat and red clover plants, the seeds 
were soaked three minutes in a 1-500 solution of mercuric chlor- 
ide, washed in sterile distilled water three times and then planted 
in sterile agar plates. By this means the seeds were sprouted 
and those which were contaminated were discovered and rejected. 
The sprouted seeds were transferred from the plates to the flasks 
by means of the platinum needle. A block of the agar containing 
the sprouted seed was cut out and placed in the proper position 
on the medium in the flask. The flasks were then carefully ob- 
served for five days tO' insure the absence of contamination. 

As all the flasks contained growing plants no attempt was made 
to exclude the light, but neither were they placed in the direct 
sunlight. They were kept on a table about eight feet from a 
large window facing the west. All the flasks were plugged with 
non-absorbent cotton and after inoculation a cap of paraffined 
paper was placed over the mouth and held in place with a rubber 
band. While the plants did not develop rapidly the oats grew 
much faster than the clover for about three weeks, after which 
time both began to lose chlorophyl and by the end of the five 
weeks' experimental period, the majority of the plants had died. 
The oats and clover in the flasks inoculated with A. chroococcum 
(HCM) and the clover in the flasks inoculated with B. radicicola 
showed a slight gain in growth and altho far from vigorous at 
the end of the experiment were still alive and growing slowly. 

ORGANISMS USET>. 

The organisms used were A. chroococcum (HCM), A. vine- 
landii, A. heijerinckii and for the purpose of comparison, B. radi- 
cicola isolated from the nodules of sweet clover. The latter were 
isolated and purifled especially for this series, and introduced to 
compare the effects of symbiotic and non-symbiotic organisms on 
the growth of the plant used. The results secured with it, how- 
ever, were of no great significance. 



38 



After the bacteria had remained midisturbed in the flasks for 
five weeks, transfers were made directly from the flasks into 50 
CO. of the nitrogen-free dextrose solution, incubated for three 
weeks, and the nitrogen fixed determined in the usual manner. 
The total amount of nitrogen fixed by the bacteria themselves, as 
well as the amount fixed by the bacteria but due to the stimulative 
action of the plants on the bacterial activities, is shown in table 
VII. There was a stimulation of the nitrogen fixing power of the 
organisms due to the presence of a growing plant, especially no- 
ticeable in the case of A. vinelandii and A. chroococcum (HCM) 
and to some extent in the case of A. heijerinckii. A. vinelandii 
was stimulated thruout the entire series except when grown in 
sand in the presence of the algae. The oats and algae showed no 

TAB'LE Vn-THE EFFElOT OF' GEIOWKSTG' PLlAW^T'S; ON THIE NITROGEN FIXING 
FOWE'Ri OF PiU.RE' ICUXTUIRIES: 



Inoculum 



Medium 



Plant Used 



(a) 



(b) 



Nitrogen Fixed in Mgs. 



(Av) 



Z-5 



Algae agar 



AJgae 

Vinelandii 

Vinelandii 

Vimlandii 

Vinelandii 

Vinelandii 

Vinelandii 

Vinelandii 

Vinelandii 

Olirooc'occmn (HCM) 

Chroococcum (HCM) 

Ohroococcum (HOM.) ._- 
Chroococcum (HOM) ..- 
Chroococcum (HCM) ..- 
Cbroococcum (HCM) .— 
Chroococcum (HOM) _.- 
Chroococcum (HCM) .._ 

Beijerinckii 

Beijerinckii 

Beijerinckii 

Beijerinckii 

Beijerinckii 

Beijennckii 

Beijerinckii 

Beijerinckii 

B. rad 
B. rad. 
B:. rad 
B. rad 
rad.. 



check 



sand check 



agar. 
sand- 
agar- 
agar- 



clo. 
clo. 

ClO'. 
ClO'. 

clo. 



rad., S. clo. 



agar 

sand 

and 

and 

agar 

sand 

agar 

agar 

igar 

sand 

sand 

sand 

agar 

sand 

agar 

agar 

Tgar 

sand 

sand 

sand 

agar 

agar 

agar 

sand 

sand 

Isand 



check 

check 

oats 

red clover- 



oats 

red clover- 



check 

check 

oatsi 

red clover. - 

algae 

oats 

red clover-- 

algae 

check 

check 

oats 

red clover- - 



oats 

red clovsr- 



oats' 

red clover- 



oats 

red clover. 



0.84 


0.84 


0.^ 


1.40 


0.98 


1.19 


1.12 


0.98 


1.05 


1.40 


1.40 


1.40 


3.66 


3. 53 


3.59 


2.10 


2.38 


2.24 


4.20 


lost 


4.20 


4.20 


4.06 


4.13 


4.48 


4.06 


4.27 


2.52 


2.80 


2.66 


O.OO 


O.OO 


O.OO 


0.28 


0.14 


0.21 


0.28 


0.44 


0.30 


1.82 


3.22 


3.52 


S.92I 


3.50 


3.71 


0.28 


0.56 


0.42 


0.28 


lost 


0.28 


3.22 


5.18 


4.20 


0.00 


0.00 


O.OO 


0.28 


0.42 


0.35 


0.14 


0.00 


0.07 


1.40 


1.40 


1.40 


O.OO 


O.OO 


0.00 


1.39 


1.39 


1.39 


1.39 


1.39 


1.39 


1.39 


1.40 


1.40 


0.14 


0.14 


.014 


0.14 


0.a4 


.014 


o.m 


0.56 


0.56 


0.42 


O.OO 


0.21 


0.14 


0.00 


0.07 


0.42 


0.50 


0.4191 



0.84 



0.84 



1.19 



1.19 



3.5t 


1.05 


2.54 


2.24 


1.05 


1.19 


3.36 


1.09 


2.31 


4.13 


1.40 


2.73 


4.27 


1.40 


2i.8'7 


1.47 


1.40 


0.07 


0.36 


0.00 


01.35 


2.52 


0.00 


2.52 


2.S7 


0.00 


2.87 


0.42 


0.21 


0.21 


0.28 


0.21 


0.07 


3.01 


0.21 


2.80 


0.07 


0.00 


o.or 


1.40 


0.00 


1.40 


1.39 


0.35 


1.04 


1.39 


0.3S 


1.04 


0.21 


0.35 





0.14 






0.14 






'o'.n 






0.07 







39 

difference when gTown on the agar and in the sand medium the 
greatest stimulation was produced by the red clover. The activi- 
ties of A. chroococcum were stimulated to the greatest extent by 
the presence of algae in both sand and agar, the oats gave a poor 
stimulation in both cases, and red clover gave good results in the 
agar but not in the sand. 

The nitrogen fixing power of A. 'beijerinckii was retarded by 
the presence of algae, but was stimulated by red clover in both 
the agar and sand. The oats stimulated this organism only when 
grown on the agar. The nitrogen fixing power of B. radicicola 
was so low thruout the experiment that the results are not con- 
sidered. 

CONCLUSIONS FROM LABORATORY STUDIES. 

1. Transfers made on a nitrogen free dextrose agar more often 
than once each week were detrimental to the nitrogen fixing power 
of azotobacter and other large celled nitrogen fixing organisms 
of the same type. 

2. Transfers made once each week into a pure sand medium 
containing some carbonaceous material were beneficial to the 
nitrogen fixing power of the azotobacter in general, but the effect 
on A. heijerincMi was detrimental. 

3. The nitrogen fixing power of A. vinelandii was stimulated 
to a marked extent when grown in large flasks for five weeks in 
the presence of red clover and oats on both agar and sand. It 
was stimulated by the presence of algae when grown on agar but 
not when grown on sand. 

The nitrogen fixing power of A. chroo'coccum was stimulated 
markedly when grown on agar for five weeks in the presence of 
growing oats and red clover, but to a less extent when grown with 
the same plants in sand. The greatest stimulation for this organ- 
ism was produced by growing it in the presence of algae in either 
sand or agar for the same period of time. 

5. The nitrogen fixing power of A. beijerinckii was stimulated 
by the presence of red clover when grown on either sand or agar, 
and by oats when grown in sand. Algae in either agar or sand 
appeared to have a depressing effect on the nitrogen fixing power 
of this organism. 

GBEENHOUSE STUDIES. 

At the conclusion of the first experiment the eight organisms 
used in the laboratory series 1, 2 and 3 were also inoculated into 
soils in pots in the greenhouse. Ground oats straw or ground 
clover hay was added to these soils and the nitrogen fixing effi- 
ciency of the organisms both in fallow soils and in the presence 
of growing oats plants determined. Three experiments were car- 
ried out in this test, as soon as the soil in which one crop had been 



40 

grown was sampled, it was immediately reseeded and another 
crop grown. Strict account was kept of the amount of nitrogen 
added in the seed and in the organic matter. The dry weight of 
the crop and the N. content as well as the nitrogen content of the 
soil was determined at the end of each experiment. 

The soil used thruout the experiment was of the type classified 
by the United States Bureau of Soils as Miami silt loam, and 
according to tests in the laboratory did not contain azotobacter 
or any similar organisms. A large amount of this soil was thor- 
oly air dried^ sieved and mixed. Ten pounds were placed in each 
of eighty glazed pots, seventy-two of which were given the fol- 
lowing treatment : Half, or thirty-six pots received an applica- 
tion of 22.68 grams ground oats straw, and the other half received 
an equivalent amount of ground red clover hay. This application 
(22.68 grams) was equivalent to a five-ton application of this ma- 
terial per acre. The ground material was thoroly incorporated in 
the soil, which was packed firmly in the pots. The pots used were 
«lazed on the inside and made tight so there was no loss by 
leaching, neither was there any drainage provided. 

METHODS OP INOCULATION. 

The inoculum used was the dextrose solution described above. 
1500 c e were placed in each of six 2 L. flasks, inoculated with the 
organism desired and incubated for seven days. Microscopic ex- 
aminations were made at the end of the incubation period to in- 
sure vigorous growth and the purity of the culture. 150 cc of 
the solution was used as the inoculum for each pot. This was 
poured over the surface of the dry soil and washed into it by the 
addition of sufficient water to bring the moisture content up to 
the optimum, in this case 25%. The pots were then weighed, 
covered with a cloth, and allowed to remain undisturbed for three 
days, in order to permit the moisture to become thoroly distrib'- 
uted thruout the soil. The pots were then arranged in the follow- 
ing manner and seeded to oats. 

Thirty grains of Early Champion oats were planted in each 
pot at each seeding. They were planted at five points. One in 
the center of the pot and the other four were arranged between 
the center and the edge at equal distances apart. Six seeds were 
planted at each place and when the plants appeared they were 
thinned out and but one plant left in each place. The discarded 
plants were allowed to remain and decay on the soil in the pot 
from which they were drawn. 

The length of the growing period was determined by the ap- 
pearance of the seed-bearing spike. This period varied slightly 
in each of the series, the first closed in sixty-three days, the second 
x'n sixty-nine days, and the third in seventy days after planting. 



41 



PLAN OP EXPERIMENT 



Pot. No. 



Treatment 



Inoculation 



1— 3 

2— i 

5h- 7 

6— 8 

9—11 

10—12 

13—15 

14—16 

17—191 

18—20 

21—23 

22—24 

25—27 

26—28-- 

29—31 

30— .32L 

33-33 

34—36 

37—39 

38-^0 

41—43 

42—44 

45-4T 

46—48 

49-51 

50—52 

5S—5S 

54—50 

57—59 

£8—60 

61—63 

62—61 

65-67 

66—68 

69—71 

70--72 

73^74 

75—76 

77—78 

79—80 



Fallow 

iCropped 

Fallow 

Oropped 

Fallow 

Cl-opped 

Fallow 

lOlropped 

Fallow 

iCtopped 

Fallow 

Cropped 

Fallow 

(Oropped 

Fallow 

iCtropped 

Fallow 

'Cropped 

Fallow 

Cropped 

FaUow 

Cropped 

Fallow 

iCtopped 

Fallow 

lOtopped 

Fallow 

Ctopped 

Fallow 

Chopped 

Fallow 

Cropped 

Fallow 

Chopped 

Fallow 

(Ctopped 

Fallow 

Cropped 

Fallow 

iCtoppeQ 



Oats straw 
Oats straw 
Olover hay 
Clover hay 
Oats straw 
Oats straw 
Clover hay 
'Clover hay 
Oats straw 
Oats straw 
'Clover hay 
Clover hay 
Oats straw 
Oats straw 
'Clover hay 
Clover hay 
Oats straw 
Oats straw 
Clover hay 
Clover hay 
Oats straw 
Oats straw 
Clover hay 
Clover hay 
Oats straw 
Oats straw 
Clover hay 
Clover hay 
Oats straw 
Oats straw 
iClover hay 
Clover hay 
Oats straw 
Oats 'straw 
Clover hay 
Clover hay 
Oats straw 
Oats straw 
Clover hay 
Clover hay 



A. chroococcum (HCIM) 

A. chroorocciim (HOM) 

A. cliriKirdcciini (HOM) 

A. clii-c)<)c(.cciiiii eH'OM) 

A. chroococcum 

A. chroococcum 

A. chroococcum 

A. chroococcum 

A. beijerinckii 

A. beijerinckii 

A. beijerinckii 

A. beijerinckii 

A. vinelandii 

A. vinelandii 

A. vinelandii 

A. vinelandii 

26 ID. 

26 'D. 

26 D. 

26 r>. 

27 D. 
27 D. 
27 D. 
27 D. 
23 D. 
22 D. 
22 D. 
22 D. 
4 D. 
4 I>. 
4 D. 
4 D. 

Mixed culture 
Mixed culture 
Mixed culture 
Mixed culture 
Cheek 
Check 
Check 
Check 



The pots were watered with tap water ahout every other day 
and were weighed weekly. The loss in weight was replaced with 
water in order to keep tlie moisture content at the optimum. The 
growth of the plants was carefully noted and recorded by means 
of photographs at different periods. The harvested plants were 
dried, weighed, and tlie total nitrogen content determined by the 
Kjeldahl method. 

At the close of each series of experiments the soils were re- 
moved from the pots, placed on a sterile oil cloth, thoroly mixed, 
sampled and returned to the original pot. The sample taken at 
this time approximated 500 grams dry weight. The pots were 
seeded again as soon as possible and the experiment continued. 
During the short period between sampling and reseeding the 
moisture content was kept at the optimum. 



42 

The preliminary analyses, showing the nitrogen content of the 
original air dried soil, and of the same soil mixed with the ground 
oats or clover are as follows : 

22.68 grs. ground oats straw contained 0.1416 grs. N. av. 6 dets. 

22.68 grs. clover hay contained 0.4153 grs. N. av. 6 dets. 

10 lbs. original soil contained 2.3494 grs. N. av. 6 dets. 

10 lbs. original soil + oats straw contained. . 2.4910 grs. N. av. 6 dets. 
10 lbs. original soil + clover hay contained. .2.7647 grs. N. av. 6 dets. 

ACTION OF DENITRIFYING BACTERIA. 

Some of the plants were very much stunted in their growth 
and an experiment was conducted to determine whether this was 
due to action by the denitrifying organisms. Samples weighing 
about six or eight grams were drawn from near the center of each 
pot by means of a sterile corkborer and placed immediately in 
sterile tubes. Sterile water was added and a soil suspension made 
from which inoculations were made into Giltay's denitrifying 
solution. The solution was incubated three weeks and the amount 
of nitrate nitrogen as well as the total nitrogen determined, the 
first by the aluminum reduction method of Potter and Snyder, 
and the second by the official method. The aluminum reduction 
was carried out by aeration, thus leaving the original solution 
available for analysis for total nitrogen. The results given in 
table VIII show that the denitrifying organisms were not the 
limiting factor in the growth of plants. Only the five soils in 
pots Nos. 29, 45, 62, 64 and 66 show any great loss in nitrogen 
and some of the pots show an actual gain in total nitrogen 
content. This gain is particularly noticeable in the soils inoc- 
ulated with A. heijerincMi, No. 26 and in the check pots. 

FABLE Vni— THE ACTIVITIES OF THE DENITRIFYING BACTERIA IN THE SOILS 
THREE WEEKS AFTER THE START OF THE EXPERIMENT 



Pot 


Nitrate N. 
mgs. 


N. mgs. 


Total N. mgs. 


Check 


Amt. denitri- 
fied 


1 


0.70 
1.05 

0.S8 
0.91 
0.90 
0.56 
0.70 
0.791 
0.70 
0.42 
0.70 
0.84 
0.86 
0.65 
0.56 


lost 

6.02 




8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 




2 

3 


7.07 


1.16 


4 " 

5 - _ __ 


5.60 

4.20 
8.12 
7.84 
8.14 
7.07 
7.07 

8.96 
6.16 
7.56 


6.51 
51.10 
8.68 
8.54 
8.93 
7.77 
7.49 

9.80 
7.02 
8.21 


1.72: 
3.13 


6 


»: 


7 




8 




9 

10 


0.46 
0.74 


11 

12 

13 . 




'1.21 


14 

15 


0.02 



43 



TABLE VIII— Continued. 



Pot 


N"itrate N. 
mgs. 


N. mgs. 


Total N. mgs. 


^, . Amt c 
Check fj 


enitri- 


16 „ . — 


1.71 
0..56 
0.90 
0.42' 
0.84 
0.S4 
0.56 
0.70 
0.78 
0.S6 
0.06 
0.58 
0.87 
0.56 

"1.54, 
0.431 
0.70 
0.56 
0.S8 
0.79 
0.S6 
0.90 
1.54 
0.63 
0.63 
0.59 
0.70 
0.98 
0.77 
1.05 
0.77 
0..5S 
O.W 
0.91 
0.81 
O.EO 
0.77 
0.49 
0.86 
1.00 
0.89 
0.86 
0.S6 
1.33 
0.91 
0.78 
1.26 
1.31 

i.v.e 

0.9S 
0.14 
0.70 
0.56 
-0.84 
0.49 
0.67 
0.87 
1.03 
0.65 
0..51 


6.10 
7.50 
7.70 
8.61 
8.40 
7.91 
9.38 


7.87 
8.12 
8.66 
9.08 
9.24 
8.751 
9.94 


8.23 
8.23 
8.23 
8.23 
8.23 
8.2s 
8.23 
8 ''3 


0.86 

o.n 


17 

18 — 


19 




20 - 




21 - .. 




22 




23 




25 I"I"I~"III""I 

26 -. - 


7.21 
6.44 
7.42 
8.04 
7.28 
5.04 
6.80 
5.32 
9.10 


7.98 
7.30 
7.48 
8.62 
8.15 
5.60 


8.23 
8.23 
8.23 
8.23 
8.23 
8.23 

8.23 

8.23 
8.23 
8.23 


0.25 
0.93 
0.75 


27 


28 


0' 08 


29 . . __ 


2 63 


30 




31 .- 


6. 86 
9.53 


1.37 


32 


33 




34 . . 


8.54 
7.56 
8.68 
7.00 
0.44 
6.58 


9.10 

8. ,54 • 

9.47 

7.86 

7.34 

8.12' 


8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 




35 




36 . - 




37 - 


37 


38 . 


89 


39 

40 - 


0.11 


41 

43 


4.90 


8.19 
5.46 


8.23 
8.23 
8.23 


0.04 
2.77 


44 - 


6.86 
2.80 
6.30 
6.16 
6.02 
7.28 


7.S4 
3.57 
7.3E 
7.. 35 
6.5-8 
8.32 


8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 


0.39 


45 - - - 


4.66 


46 - -. . 


0.88 


47 

48 . . 


0.8» 
1.65 


49 

50 




51 


7.77 
7.70 
8.12 
7.56 
6.44 
6.09 
5.40 
7.70 
5.60 


S.'S 
8.20 
8.89' 
8.05 
7.30 
7.09 
6.32 
S.56 
6.46 


8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 . . 






0.03 


53 

54 


0.18 


53 -. 


0.83 


56 


l.U 


57 

58 — 

59 

60 


1.81 
1.77 


61 






8.23 




63 .. .. . 


1.96 
5.60 
4.76 
7.28 
1.54 
7.28 
6.16 
6.72 
4.6-2 
8.40 
7.98 
6.02 
7.70 
9.80 
8.12 


2.74 
6.86 
6.07 
8.. 54 
2.52 
7.42 
6.86 
7.28 
5.46 
8.89 
8.65 
6.89 
8.75 
10.43 
8.63 


8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 
8.23 


5.49 


63 


1.37 


64 — 


2.10 


65 . . 




66 ._ .. . . ... 


5.71 


67 

68 


0.81 
137 


69 . - 


0.95 


70 

n . . . - _ 


2.77 


72 - . 




73 


1.34 


74 




75 . . .. 




76 





*'So denitrification is shown by : 



44 



PRELIMINARY TESTS FOR NITROGEN FIXATION. 

To discover the action of the bacteria in the inoculated soils 
samples were taken from the fallow pots four weeks after t': e start 
of the experiment and their total nitrogen content determined. 
Table IX shows a gain in the nitrogen content over the original 
soil and the check soils but the actual gain due to the action of 
the bacteria introduced was very slight. Organisms 22, 4 and 
the mixed cultures showed no gain whatever, and the others 
showed only a slight gain in those soils to which clover had been 

TABLE IX — THE ACTIVITY OP THE BACTERIA IN THE INOCULATED 

FALLOW SOILS AFTER FOUR WEEKS. 

Grams of Nitrogen Per 10 Pounds of Soil. 



Pot 



Gi-ams N. 



1 

2 

5 

7 

9 

11 

13 

]5 

17 

19 

21 

23 

25 

27 

29 

SI 

33 

35 

3Y 

3& 

41 

43 

45 

47 

49 

51 

5S 

55 

57 

59 

&1 

63 

65 

67 

60 

n 

Checks 

73 

74 

77 

78 



M O'o 

o 



.Set. 

. C3 O 



0643 
0643 

8942 
8304 
0643 
2281 
3411 
9581 
2281 
2281 
2134 
0219 
2281 
''239 
7666 
2772 
2f281 
0613 
8942 
8942 
2281 
1929 
9581 
7665 
8409 
9047 
7027 
5431 
8409 
8089 
5431 
.5750 
9047 
7451 
2239 



.5536 
0043 
93-66 
0643 



Average four checks- 



lost 
3.2281 
3.8942 
3.9581 
3.2281 
3.2281 
4.0S96 
4.0S57 
3.2281 
3.06i!3 
4.0219 
3.9'561 
3.2558 
3.2239 
4. 0857 
4.0219 
3.2281 
3.1920 
3.8304 
3.8304 
e.25S8 
3.228.1 
3.9561 
3.8304 
2.8409 
2.7679 
3.8942 
3.6069 
2.9047 
2.7132 
3.5752 
3.6069 
2.8408 
a. 7451 
3.1320 
3.443a 

2.8089' 
3.0324 
2. 8089 
3.2239 



3.0643 
3.1462 

3.8942 
3.8942 
3.1462 
3.2281 
4.2203 
4.0219 
3.2281 
3.1462 
4.1176 
3.9900 
3.2419 
3.2239 
3.9261 
4.1490 
3.228] 
3.1281 
3.8623 
3.862f 
3.2419 
3.210:' 
3.9581 
3.7984 
2.8409 
2.8393 
3.798! 
3.5750 
2.872? 
2.7610 
3.5590 
3.5909 
2.8727 
2.745] 
3.2079 
3.5250 

2.6S12 
3.0483 
2.8727 
3.H4] 



2.4910 


0.5533 


2.4910 


0.5552 


2.7647 


1.1295 


2.7647 


1.1295 


2.4910 


0.6552 


2.4910 


0.7371 


2.7647 


1.4556 


2.7647 


1.2572 


2.4910 


0.7371 


2.49110 


0.6552 


2.7647 


1.3529 


2.7647 


1.2258 


2.4910 


0.7509 


2.4910 


0.7329 


2.7647 


1.1614 


2.7647 


1.3843 


2.4910 


0.7371 


2.4910 


0.6871 


2.7647 


1.0976 


2.7647 


1.0976 


2.4910 


0.7509 


2.4910 


0.7109 


2.7647 


1.1934 


2.7647 


1.0337 


2.4910 


0.8499 


2.4910 


0.3453 


2.7647 


1.0837 


2.764.7 


O.8103 


2.4910 


0.S818 


2.4910 


0.2700 


2,7647 


0.7943 


2.7647 


0.S262 


2.4910 


0.3817 


2.4910 


0.2541 


2.7647 


0.4432 


2.7647 


0.7603 


2.3494 


0.3318 


2.3404 


0.6989 


2.3494 


0.5233 


2.3494 


0.7947 



0.7288 
0.7288 
1.0025 
1.0025 
0.7288 
0.7288 
1.0025 
1.O025 
0.7288 
0.T288 
1.0025 
1.O025 
0.7288 
0.7288 
0.0025 
1.002s 
0.7288 
0.7288 
0.0025 
1.0025 
0.7288 
0.7288 
1.0025 
1.0025 
0.7288 
0.7288 
1.0025 
1.0025 
0.7288 
0.7288 
1.0025 
1.0025 
0.7288 
0.7288 
1.0025 
1.0025 



§0. 

5 cS <u 

o 



0.1270 

0.1270 

0.0083 
0.4531 
0.2547 
0.0083 

0.3504 
0.2228 
0.0221 
0.0041 
0.1569 
0.3818 
O.00S3 

0.0951 
0.0961 
0.0121 

0.1909 
0.0312 



0.0312 



.0.5672 + N in oats 0.1'. 16=--0. 7288 
.0.5872' + N in clo. 0.4153^1. 0025 



45 

added. This difference may have been due to variation in the rate 
of decomposition between the clover and the straw. 

At the end of the three growing periods the soil in each pot 
was sampled and the total nitrogen content of both the soil and 
the entire crop determined. The amount of nitrogen found in 
the determinations and its relation to the total amount due to 
the bacterial activities is given in three separate tables, one for 
each growing period. From these complete tables three condensed 
tables have been made as follows : For the first growing period, 
table X, for the second growing period, table XI, for the third 
growing period, table XII, and a recapitulation table XIII. 



TABLE X— THE NITROGEN FIXED BY BACTERIA-FIRST PERIOD 
(Condensed from Appendix Table I.) 





Treatment 


Bact. Inoculum 
Used 


Grams N. per 


10 Pound 


s Soil 


■p. o 
3 a 
P 


o 

2; 


" ■« > ft 

_c g o o 

'" CS S Cj 

2: 




2; 


1— 3— 


P oats 

C oats 

F clover — 

C clover 

F oats 

O oats 

F clover 

O clover .„ 

P oats 

C oats 

P clover .. 

O clover 

P oats 

C oats — _ 

P clover . 

O clover 

P oats 

O oats 

P clover 

O clover 

F oats 

O oats . 

P clover 

O clover 

P oats 

O oats 

P clover _- 

C clover 

F oats 

lO oats 

P clovsr 

O clover 

P oats 

C oats 

P clover 

C clover _— 

P. nothing _— - 

O. nothing 

P nothing 

C nothing 


A. chrooe. (HClM) — 
-4. chrooe. (HCM) — 
A. chrooe. (HCM) — 
A. chrooe. (HCM)— . 
A. chrooe. 


3.3499 
2.9S83 
4.3058 
4.1808 
3.2210 
3.5380 
4.5726 
4.4928 
5.1701 
5.2867 
4.4S94 
4.5801 
3.9267 
5.1120 
2.4572 
4.2388 
3.4887 
3.5170 
4.4823 
4.5722 
3.6458 
3.5720 
4.5023 
4.3114 
3.4101 
3.5823 
4.4001 
4.3114 
3.6065 
3.50C6 
4.4630 
4.30-23 
3.3865 
3.60S8 
4.3787 
4.4720 
3.5942 
3.5085 
3.3002 
3.5403 


3.3833 
2.9670 
4.3488 
4.-2684 
3. -2582 
3.5808 
4.6183 
4.5787 
5.2218 
5.28-24 
4.4827 
4.6613 
3.95159 
5.1794 
2.4817 
4.3457 
3.5235 
0.5133 
4.5-271 
4.6833 
3.6822 
3.6217 
4.5473 
^4196 
3.4442 
3.5519 
4.4441 
4.3866 
3.6423 
3.. 5323 
4.5076 
4.3965 
3.4203 
3.60-21 
4.4123 
4.5794 
3.6301 
3.. 5689' 
3.3332 
3.6097 


3.6232 
3.7309 
3.8963 
4.0046 
3.6232 
3.7309 
3.8963 
4.O046 
3.6232 
3.7309 
3.8963 
4.0046 
3.6232 
3.7309 
3.8063 
4.0046 
3.6232 
3.7309 
3.8963 
4.0016 
3.6232 
3.7309 
3.8963 
4.0046 
3.6232 
3.7-209 
3.8963 
4.0046 
3.62.32 
3.7309 
3.8963 
4.0046 
3.6232 
3.7309 
3.8963 
4.0046 




2— 4. 




5— 7- 

6— S-. 
&-11.- 

I(t— 1'^ 


0.4525 
0.-2638 


13-15-- 
14—16- 
17—19 - 


A. chrooe. 

A. chrooe. 

A. beyer. .. -. - 


0.7220 
0.5741 
1..5966 


18—20— 


A. beyer. .. 


1.5515 


21—23— 

')9 — Oi , 


A. teyer. 

A. beyer.^ .._ 


0.5884 
0.6567 


25—27- 


A. vine. - - 


0.34-27 


26—28- 


A. vine. _. 


1.4485 


29^—31 


A. vine. 




30—32- 


A. vine. -, 


0.3411 


33—35 - 


No. 26 . 




34—36 - 


No. 26 




37— 3&- 


No. 26 - 


0.6808 


3S^}0.. 
41—43- 
42-44-- 


No. 26 

No 27 

No. 27 


0.6787 
O.O590 


45-^7- 
46—48 - 


No. 27 

No. 27 


0.6510 
0.4149 


49—51- 


No. 22 .- 




50—52— 
53—55- 


No. 22 

No. 22 


0.5478 


.S4-56- 

57— 59- 
58—60 


I.o. 22 

No' 4-IZ-II-II-I." 

No. 4 


0.3820 
0.0193 


61-63- 


6113 


62-61 


No 4 - 


3919 


65—67- 
66-68- 


Mixed culture 

Mixed culturs 

Mixed culture 

Mixed culturs 

Check - 




6»-7l- 

70—72— 
73—74 


0.5160 
0.5748 


7.5—76- 


Cheek 






77— 7S- 
79^80- 


Check 

Check 








46 



FIRST GROWING PERIOD. 



The determinations for this period are shown in appendix 
table I and in condensed table X. As indicated by table IX, 
there was a steady increase in the total amount of nitrogen fixed 
in all the soils. This increase is still more marked if the last 
columns of tables IX and X are compared. The bacteria were 
increasing-ly active in fixing- the free atmospheric nitrogen and in 
practically every case the total amount fixed due to the bacterial 
solution was more than doubled during- the latter five weeks of 
this series. 

These activities may be divided into two classes, as the bacteria 
were more markedly affected by the presence of clover hay or 
of oats straw. In the first class A. ckroococcmn, A. chroococcum 
{HCM), No. 26, No. 22 and the Mixed Culture stood out prom- 
inently. None of these four organisms showed any fixation due 
to the presence of the decaying oats straw, but they did show ap- 
preciable g'ains due to the presence of the clover hay. The pres- 
ence of the oats straw had apparently either inhibited the activi- 
ties of the organisms or increased the activities of the other forms 
that are incapable of fixing nitrogen for their own use and have 
utilized that fixed by the inoculating organisms. Organisms 4 
and 27 showed a decided stimulation due to the clover hay and 
were able to utilize the oats straw as a source of energy, 

A heijerincki and A. vinelmidii were more markedly affected by 
the presence of oats straw. The stimulation of the activities of 
the former due to the presence of the decaying clover was parallel 
to that of the other organisms, and in addition the presence of the 
decaying oats straw stimulated its nitrogen fixing powers to over 
250% of that of any other organism in the series with the single 
exception of A. vinelandii. On the other hand, A. vinelandii, 
while showing a marked stimulation due to the presence of the 
oats, also showed that the clover hay affected its activities much 
the same as the oats straw affected the other organisms, that is, 
the presence of the decaying clover hay in the cropped soils, de- 
creased its nitrogen-fixing power, and in the fallow soils, com- 
pletely inhibited it. 

SECOND GROWING PERIOD. 

The results for this period are shown in appendix table II, the 
more important parts of which are repeated in condensed table 
XI. In comparison with the first gTowing- period the results for 
the second period are decidedly lower thruout the second series. 
Not only are the total amounts of nitrogen found lower, but also 
the total amount of dry matter produced in the crop, indicating 
a possible direct relation between bacterial action and crop yields. 
These low results are explained by the fact that this series as 



47 

grown during' the hottest part of the summer, the pots being 
planted in the latter part of June and harvested during the earl- 
ier part of August. The results confirm those given in Table X 
except that in this series the only organism stimulated by the 
presence of the decaying oats straw was organism 27 in the 
cropped pots. Each of the inoculated organisms showed a direct 
stimulation due to the presence of the clover hay. 

The organisms may be divided into two classes according as 
their activities are stimulated or retarded by the presence of 

TIAJBCLE XI— THE NinHiOO'EJST MXEID BY BiAlCITIElRlLA— iSECIOiND' PERIOD. 
('Oondensed from appendix Table 2.) 



Dupli- 
cate 
Pots 



Tieatment 



Bacterial inoculum 
used. 



Grams Nitrogen per 10 lbs. soil. 



g > 2 
sou 

£ Z't 

tic 2 
2g^ 



•^2 



1— 3— 

2— 4._ 
&— 7... 
&- 8... 
9—11.. 

10-412... 
13—15.. 
14^16... 
17—19... 
IS— 20.. 
21—23.. 
22—24.. 
25—27.- 
2&— 28„ 
291—31... 
iO— 32.. 
33—35.. 
34—36.. 
87—39.. 
38—40.. 
41—13.. 
42—44-. 
45—47-. 
46^48.. 
49^51- 
50^52.. 
53—55.. 
54—50- 
57—59.. 
5S— 60.- 
61—68-. 
62^64-. 
eo'— 67-. 
66— 68-. 
69—71- 
70—72- 
73—74-. 
75-76-. 
77—78-. 
79—80- 



oats 

oats -. 

clover 

clover 

oats 

oats 

clover 

clover 

oats 

oats 

clover 

clover 

oats 

oats 

clover 

clover -.... 

oats 

oats 

clover 

clover 

oats 

oats 

clover 

clover 

oats 

oats 

clover 

clover 

oats 

oats 

clover 

clover 

oats 

oats 

clover 

clover 

nothing- ... 

nothing 

nothing 



A. chroocoecum (HCIM). 
A. chroocoecum (HiOM). 
A. chrooeoceum (HICM). 
.4.. chroocoecum (HIOM). 

A. chroocoecum 

A. chrooeoceum 

A. chroocoecum 

A. chroocoecum 

A. Beijerinckii 

A. Beijerinckii 

A. Beijerinckii 

.4. Beijerinckii 

A. vinelandii 

A. vinelandii 

A. vinelandii 

A. vinelandii 

No. 26 

No. 26 

No. 26 

No. 26 

No. 27 

No. 27 

No. 27 

No. 27 

No. 22 

No. 22 

No. 22 

No. 22 

No. 4 

No. 4 

No. 4 

No. 4 

Mixed culture 

Mixed culture 

Mixed culture 

Mixed culture 

Check 

Cheek 

Check 



nothing ... Oheck 



2.7340 


3.0128 


2.8218 


3.0916 


3.3205 


3.6592 


3.3123 


3.6402 


3.6867 


2.9607 


e. 54791 


2.7947 


3.5242! 


3.8836 


3.1813 


3.4901 


2.7155 


2.9924 


2.6134 


2.8626 


3.3343 


3.6743 


3.21681 


3.5857 


2.8239 


3.1115 


2.6936 


2.9763 


3.3205 


3.6591 


3.4143 


3.6822 


2.7547 


3.0350 


2.8912 


3.1730 


3.4921 


3.8492 


3.0648 


3.3747 


2.7015 


2.9770 


8.O508 


3.3429 


3. 4487 


3.8004 


3.5089 


3.8613 


2.7083 


2.9845 


2.8804 


3.170O 


3.3977 


3.7442 


3.2667 


3.5925' 


2.6146 


2.8812 


2.8246 


3.1262 


3.3998 


3.7465 


3.1740 


3.4913 


2.5210 


2.7781 


2.6935 


2.9S10 


3.21396 


3.6031 


3.38521 


3.7159 


2.8835 


3.1115 


2.7518 


3.0.537 


2.9758 


3.2798 


2.7736 


3.0364 



3.3370 




3.1866 




3.6107 
3.4603 
3.3370 


O.0485 
0.1799 


3.1866 
3.6107 
3.4603 
3.3370 


'"0^2729 
0.0298 


3.1866 




3.6107 
3.4603 
3.3370 
3.1866 
3.6107 
3.4603 
3.3370 


O.O630 
0.1254 

"o.0384 
0.1719 


3.1866 




3.6107 
3.4603 


0.2385 


3.3370 




3.1860 
3.6107 
3.4603 
3.3370 
3.1866 


0.1503 
0.1897 
0.4010 


3.6107 
3.4603 
3.3370' 
3 I860 


0.1335 
0.1322 


3.6107 
3.4003 
3.3370 
3.1866 


0.1358 
0.0310 


3.6107 




3.4603 


0.2550 









48 

growing plants such as clover. In the first class are included A. 
chroococcum {RCM) , A. heijerinckii, A. vinelandii, No. 27 and 
the mixed cultures. The first three organisms have had their 
nitrogen-fixing powers stimulated by the presence of the plants 
in practically the same ratio and have fixed similar amounts in 
both the fallow and cropped soil. A. h&ijerincJdi showed the 
highest fixation of any of the eight for this series. The mixed 
cultures showed no fixation whatever in the fallow soils, but 
quite an appreciable amount in the cropped soils. No. 26, on 
the contrary, fixed an appreciable amount of nitrogen in the 
fallow soils but none at all in the presence of the growing oats 
plants. A. chroococcum. and No. 4 showed the same stimulation 
under practically the same conditions, namely, that they possess 
a greater nitrogen-fixing power in the presence of decaying clover 
if no crop is grown upon the soil, while No. 22 was apparently 
neither stimulated nor retarded by either fallow or cropped con- 
ditions, but was affected by the presence of the decaying oats 
straw. 

THIRD GROWING PERIOD. 

The results for this period are shown in appendix table III 
and condensed table XII. The total nitrogen content of the soil 
according to tables IX, X and XI, increased steadily through- 

TABLE XII— THE NITROGEN FIXATION BY BACTERIA— THIRD PERIOD 
(Condensed from Appendix Table III). 







Bacterial Inoculum 


Grams Nitrogen per 10 Pounds Soil 






+ ,^ 


-Sll 


>> 






Treatment 




Used 


a 


l£-^ 


.^ O'S 


"S 53 


«§ 










3 

o 


in s 
amt. 
mov 
crop 


.s-^l 






■^ 












^•^ 




fi; 








^ 


1 ^ 


;z; 


^ 


< 


1 


F oats 


A. 


chrooc. (HCM) 


2.56-2:6 


2.8618 


2.8502 


0.0146 




R 


F oats 


A. 


chrooc. (HCM) 


2.7342 


3.3166 


2.8502 


0.4063 


0.2405 




'O oats 

O oats 


i 


chrooc. (HCM) 


2. 3643 


2,9647 


3.3284 






4 


A. 


chrooc. (HCM) 


2.1930 


2.6754 


3.32S4 






5 


F clover _ 


A. 


chrooc. (HCM) 


2.0255 


3.5486 


3.1239 


0.4247 





7 


F clover 


A. 


chrooc. (HCM) 


2.8274 


3.4296 


3.1239 


0.3057 


0.3652 


6 


O clover . 


A. 


chrooc. (HCM) 


S.2852 


4.0691 


3.6021 


0.4670 




S 


O clover 


A. 


chrooc. (HCM) 


2.S688 


3.16142 


3.6021 


0.0121 


0.2395 


9 


F oats — - 


A. 


Chroococcum.. 


21.7489 


3.3344 


2.8502 


0.4842 





n 


F oats _ 


A. 


Chroococcum.. 


2.3103 


2.8024 


2.8502 




0.2421 


10 


oats 


A. 


Chroococcum.. 


2.3652 


2.9149 


3.3284 






12 


O oats 


A. 


Chroococcum.. 


2.S7S5 


2.9537 


3.3284 






13 


F clover 


A. 


Chroococcum... 


2.8928 


3.5089 


3.1289 


0.3850 


13 


F clover 


A. 


Chroococcum.. 


3.1540 


3.8265 


3.1239 


0.7026 


0.5438 


14 


O clover 


A 


Chroococcum.. 


2.8489 


3.5649 


3.6021 






Ifi 


'O Clover 


A 


Chroococcum. 


2.7501 


3.4326 


3.6021 






17 


F oats 


A. 


beijerinckii 


2.5789 


3.1279 


2.8502 


0.2777 





19 


F oats 


A. 


beijerinckii 


2.4S05 


3.0088 


2.8502 


0.1586 


0.218B 


18 




4 


heijerinckii 

beijerinckii 


2.2943 


2.8469 


3.3284 






20 


O oats 


A. 


2.07317 


2.5764 


3.3284 








21 


F clover 


A. 


beijerinckii 


3.5208 


4.2705' 


3.1239 


1.1466 




2i3' 


F clover .. 


A. 


beijerinckii 


S.0S68 


3.6836 


3.1239 


0.5597 


0.8532 



49 



TABLE XII— Continued 





Treatment 


Bacterial Inoculum 
Used 


Grams Nitrogen Per 10 Pounds Soil 




-0 


-a 1 -° 
5i ^"S-- 








w 






3 


■" tj > a. 
■-Ill 


.5 c c 




^t 


O 

a. 










. Jl 


>-° 






12 


2i 


^ 


z 


< 


22 


clover 


A. beijerinckii 


2.7429' 


3.4256 


3.6021 







24 


clover 


A. beijerinckii 


2.7694 


3.4584 


3.6021 






Wi 


F oats 


A. vinelandii 

A. vinelandii 

A. vinchindii 


2.5431 
2.447S 
2.3917 
2 3491 


3.0847 
2.9601 
2.9781 
2.9494 


2.8502 
2.8502 
3.3284 
3.3284 


0.2315 
0.1189 




97 


F oata - 


0.1767 


26 


oats 




28 


oats 

F clover 






29 


A. rinrliiniHi 


S.0172 


3.6598 


3.1289 


0.5359 




31 


F clover 


A. r'nuUmiUi 


3.0711 


3.7252 


3.1239 


0.6013 


0.5686 


80 


clover 


A. rhir'KiniHi 


2.7429 


3.4284 


3.6021 







32 


clover 


A. rhicUindii 


2.7363 


3.3959 


3.6021 







33 


F oats 


No. 26 


2.4609 


2.9850 


2.80O2 


0.1348 




S3 


F oats 


No. 26 


2.3823 


2.8887 


2.8502 


0.0385 


0.0862 


34 


oats 

oats 


No. 26 

]So. 20 , 

^*b. 25 


2.4848 
2.3321 

2.9S95 


3.0673 
2.9746 
3.6162 


3.3284 

3.3284 
3.1239 






.% 


' 0^4923 




m 


F clover 


, 


39 


F clover 


No. 26 


2.0164 


2.4459 


3.1239 




0.2463 


38 


lO clover 


No. 26 


2.7749 


S.4477 


3.6021 






40 


'O clover 


No. 26 


2.8877 


3.5&S8 


8.6021 







41 


F oats 


No. 27 

No. 27 


2.4478 
2.4871 


2.9691 
3.0169 


2.8502 

2.8502 


0.1189 
0.1667 




43 


F Oatsi 


0.1428 


4f1 


oats _, 

oats 


No. 27 .. 


2.4583 
2.4917 
2.8143 


3.0736 
3.1595 
S.4137 


3.3284 
3.3284 
3.1289 






44 


No. 27 . 

No. 27 






45 


F clover 


0.2898 




47" 


F clover 


No. 27 


3.0564 


3.7074 


3.1239 


0.5835 


0.4366 


46 


clover „„ 


No. 27 . 


3.0181 


3.7421 


3.6021 


0.1400 





48 


iO clover 


No. 27 


2.8621 


3.5807 


3.6021 




0.0700 


49 


F oats 


No. 22 


2.4961 


3.0278 


2.8502 


0.1776 





51 


F oats 


No. -221 


2.2186 


2.6911 


2.8502 




0.0888 


50 


oats 

oats . 


No 221 . 


2.5580 
2.4451 


3.1751 

3.0758 


3.32S4 

3.3284 







5?; 


No. 22 




53 


F clover 


No. 22! 


2.7230 


3.3029 


3.1239 


0.1790 





55 


F clover 


No. 23 


2.8667 


3.4773 


3.1239 


0.3584 


0.2662 


54 


clover 

clover _. 

F oats 


No. 23 

No. 23 


2.7893 
2.6236 
2.3430 


3.5060 
S.29S3 
2.8321 


3.6021 
3.6021 
2.8502 






5fl 






57- 


No. 4 






59 


F oats 

'O oats 


No. 4 . 


2.4094 
2.3170 
2.60S7 


2.9126 
2.8086 
3.3108 


2.8502 
3.3284 
3.3284 


0.0624 


0.0312 


58 


No. 4 

No. 4 




60 


lO oats 




_. 


fil 


F clover 

F clover 

clover 


No. 4 


3.0173 

2.8208 

2.8877 


3.6599 
3.4216 
3.6172 


3.1239 
3.1239 
3.6021 


0.5860 
0.2977 
0.0151 




m 


No. 4.^ 


0.416S 


62 


No. i 





M 


a Clover 

F oats 


No. i_ 


2.8224 
2.3496 


3.5098 
2.8501 


3.6021 




0.0076 


65 


Mixed culture 


2.8502' 




67 


F oats 


Mixed culture 


2.3234 


2.8183 


2.8502 





m 


oats - 


Mixed ciiltiire 


2.4381 


3.0435 


3.3284 




fiS 


lO oatsi - . 


Mixed culture 


2.3586 


2.89S7 


3.3284 




69 


F clover 


Mixed culture 


3.0761 


3.7313 


3.1239 


0.6074 





71 


F clover 


Mixed culture 


2.7831 


3.3759 


3.1239 


0.2520 


0.4297 


70 






2.8366 


3.5867 


3.6021 






72 


C clover . 


Mixed ci.lture 


3.0306 


3.7664 


3.6021 


0.1643 


0.0822 


73 


F nothing- 


Check 


2.3216 


2.8161 


* 


* 


* 


74 


F. nothing 


Check 


2.0944 


2.5405 


* 


* 


* 


77 


F nothing 


Check 


2.1074 


2.5568 


* 


* 




78 


F nothing 


Check 


2.4085 


2.9215 






* 


75 


nothing 


Check 


2.7760 


3.3649 


!t 


t 


t 


7fi 


nothing 


Cheek 


2.5375 


3.1041 


+ 


+ 


+ 


79 


nothing 


Check 


2.4911 


3.1522 


t 


t 


t 


80 


nothing 


Cfteek 


2.4901 


3.1362 t ' t t 



•Average four fallow checks 2.7086. 
■fiAverage four cropped checks 3.1868. 



50 

out the first growing period, declined somewhat during the 
second, and according to table XII, there was a pronounced ten- 
dency to increase again during the third period. The crop re- 
sponse of this last period of growth confirms the results of the 
determinations, the amount of dry matter produced being prac- 
tically midway between the production of the first and second 
growing periods. Figs. 1-6, which show the growth of oats in rep- 
resentative pots for the three periods, show that the first crop 
when ready to harvest was in the majority of cases leafy and 
heavy and showed a decided tendency to lodge ; the second crop 
in the same stage of growth was somewhat dwarfed in appearance 
and with no indication of leafiness or weakness of stem ; the third 
crop, while not as heavy as the first, showed all of its characteris- 
tics except that as a whole the production was more uniform and 
did not show the variation in the total dry weight of the harvested 
crop. The bacterial activities, which are plotted in the tables 
shown in fig. 7, varied in the same proportion as the crop re- 
sponse of the treated soils, being practically parallel with the 
production of the dried weight cf the crop. The activities in- 
creased during the first growing period, declined thruout 
the second, but increased again during the third. The discus- 
sion of the third and last period of growth will be a combina- 
tion of the activities of the inoculated bacteria as discussed in 
the first and second growing periods. 

The last column in table XII indicates that each inoculated 
bacterial culture acted without exception in the same general 
manner instead of showing the expected variations. All of the 
inoculated bacteria fixed greater amounts of nitrogen in the 
soils to w^hich clover hay was added as organic matter than in 
soils that were treated with the same amount of oats straw, and 
the growing crop on these soils reduced the nitrogen-fixing power 
of each and every one of these organisms. The activities of any 
one of the eight organisms used during the third period of growth 
would be an accurate measure for the activities of any of the 
others, a fact not even indicated in the other periods of growth. 

Conclusion: Table XIII, recapitulating tables X, XI and 
XII, shows that inoculation, especially in fallow soils to which 
clover hay or oats straw was added, is not only possible but 
practical. The amounts of nitrogen shown in these tables are 
the actual amounts fixed by the organisms in ten pounds of soil 
and if these amounts are calculated on a 2,000,000 pound acre 
basis, the result is distinctly profitable. With proper soil condi- 
tions the greenhouse experiments can be duplicated in the field. 

All of the organisms have shown an appreciable fixation of 
nitrogen but A. beijerinckii and A. vinelandii have been de- 
cidedly the most active. This finding confirms the suggestion of 



51 



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52 




Fio. 1. Oats at end of first growing period, immediately before harvest; in pots 2, 6, 10. 14, IS, 22 

26, 30, 24, 38 



\U 










Fig. 2. Oats at end of first growing period, mmediately before harvest; pots 44, 48, 52, 56, 60, 64, 

68, 72, 76, 80 




Fig 3. Oats at end of second growing period, immediately before harvest; pots 2, 6, 10, 14, 18, 22 , 

26 30, 34, 38 




Fig 4 Oats at end of second growing period, immediately before Harvest; pots 42, 46, 50, 54, 58, 

62, 66, 70, 75, 79 



\ -v. 



1/7' \ 



\\\ 







r--^r 



IG. 5. Oats at end of third growing period, mmediatelv before harvest; pots 4, 8, 12, 16, 20 24. 

28, 32. 36, 40 




Fig. 6. Oats at end of third growing period, immediately before harvest; pots 44 48, 52, 56 60 

64, 68, 72, 76, 80 



54 















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LEGEND. 
Oata added as monure 

yjote HepffaHooJ 

OqH adaea as manure 

pais kept cmpped-- «- 

Oovei-Qddsd as manure 

oofs kept fallow 

C/over added os monure 

;OoM HepfcmppecJ 
















































































tr 


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Fig. 7. This graph shows the variation in bacterial activity in the different growing periods 



55 

Lipman and Brown (41) by proving definitely that these organ- 
isms are capable of being profitably inoculated into field soils, 
provided that organic matter, carrying a sufficient amount of 
nitrogen as a stimulus, is supplied. 

Simimary. 

1. When three crops of oats were grown continuously on this 
soil the nitrogen content of the soil increased during the first 
cropping period, decreased during the second, and increased 
slightly again during the third. 

2. The nitrogen-fixing powers of the bacteria and the crop 
response were parallel with the total nitrogen content of the soil. 

3. The nitrogen-fixing powers of some types of azotobacter 
and other large celled organisms of the same general character, 
were stimulated to a greater extent by the presence of decaying 
clover hay than of decaying oats straw. 

4. The nitrogen-fixing powers of A. heijerinckii and A. 
vinelandii were stimulated to a greater extent by decaying oats 
straw than by clover hay, especially during the earlier stages of 
decomposition. , 

5. The nitrogen-fixing powers of the azotobacter and other 
large celled organisms of the same general type eventually be- 
came greater in fallow than in cropped soils. 

6. The non-symbiotic nitrogen-fixing organisms of the azoto- 
bacter group were all eventually influenced in their activities in 
the same manner and by the same materials. 

7. Soils may be profitably inoculated by azotobacter and 
other large celled organisms of the same type, the best effects 
being secured in this work by an inoculation with A. heijerinckii 
or A. vinelandii. 

8. The conditions necessary for the greatest fixation are : 
Good environmental factors such as tillage, drainage, etc. ; the 
presence of a rapidly decaying organic matter carrying a small 
nitrogen content, and freedom from growing plants. 

ACID EXTRACT, AMINO, NON-PROTEIN AND POLYPEP- 
TID NITROGEN CONTENT OF THE POT SOILS. 

Introduction : The nitrogen of the soil is found in many com- 
plex combinations, in the determination of which the Bureau of 
Soils has isolated a large number of nitrogenous compounds and 
many different forms have been discovered. In investigating 
methods for the determination of amino acids and nitrates in a 
limed and unlimed soil, both with and -without heavy applications 
of manures. Potter and Snyder (53) have found that they could 
accurately measure the 'amino nitrogen by a modification of the 
method devised by Kober and Sugiura (32). They discovered 



56 

no tendency for the amino acid to accumulate under the condi- 
tions of the experiment. Accordingly in the present investiga- 
tion determinations were made of the acid extract, non-protein, 
amino, and polypeptid nitrog^en of some of the soils inoculated 
with the azotobacter cultures used in the g:reenhouse experi- 
ments, in order to prove this point and also to discover if the 
bacterial action had any effect on the accumulation or disap- 
pearance of these nitrogenous forms. 

Soils used: Only the three soils inoculated with A. chroo- 
coccum, A. heijerinckii and A. vinelandii were analyzed. 

METHODS. 

Acid extract : Place 166 gr. of air dried soil on a wetted 
double filter paper in a Buchner funnel and extract with 600 e. c. 
of a 1% HCl solution using g-entle suction. Keep the soil barely 
covered with the solution and when extracted, wash with 200 to 
300 c. c. of pure distilled water. Dry as quickly as possible, 
and determine the nitrogen content of the filtrate jjy the official 
salicylic acid method. 

Alkali extract: The non-protein, amino, and polypeptid ni- 
trogen determinations are based on the amounts extracted by a 
1.5% NaOH solution. Shake 150 gr. of the air dried acid ex- 
tracted soil with 600 c. c. of the NaOH solution and centrifuge to 
a clear solution. At least 210 c. c. cf the clear solution must be 
obtained. 

Non-protein nitrogen : Pipette off 25 c. c. of tbe alkali extract, 
neutralize Avith a sulphuric acid solution and add sufficient tri- 
chloracetic acid to make a 2.5% solution. To do this use 4.3 c. e., 
of a 1 3/10 N. H2SO4 solution and 0.75 c. c. of a saturated tri- 
chloracetic solution. This method precipitates the proteins which 
are removed by filtering. Pipette 10 c. c. of the clear filtrate 
into large test tubes, add a couple glass beads, 2 drops of a 5% 
CuSO^ solution, 1 c. c. C. P. HoSO^, and approximately 1 gr. 
C P. potassium sulfate. Digest and distil as in the regular 
Kjeldahl method determining the ammonia colorimetrically. 

Amino acid nitrogen : Pipette 80 c. c. of the alkali extract into 
100 €. c. measuring flasks, neutralize with strong HCl until 
neutral to litmus, add 7 c. c. saturated lead acetate solution, fill 
the flask to the mark with concentrated NH^OH and shake vig- 
orouly. Allow to settle for a few minutes then pass through 
double filter, using gentle suction and obtain at least 80 c. e. of 
the filtrate. Measure oft' 75 c. c. of this filtrate, add 25 c. c. 
saturated Ba(0H)2 and phenolphthalein as indicator and distill 
over steam bath under reduced pressure until there remains a 
volume of about 25 or 30 c. c. It is important that the reaction 
of the solution throughout this distillation should be at all times 



57 

alkaline. Discard the distillate, wash residue into 100 c. c. gradu- 
ate, cool, make up to 75 c. c, filter quickly to remove all car- 
bonates, pipette 50 c. c. into 100 c. c. measuring flasks, make ap- 
proximately neutral with N/10 HCl and add 40 c. c. of buffer 
solution, stopper tig'htly and keep in cool place, if possible, on 
ice. (The buffer is made by dissolving- 0.2 gr. molecules of boric 
acid in water, adding 100 c. c. of COo free N/10 NaOH solution 
and making up to 1000 c. c. with pure COo free water. Three 
volumes of this mixed with one volume of 0/1/N HCl makes the 
desired solution.) 

Use pure water as cold as possible to prepare fresh the fol- 
lowing solution: Place 10-20% copper chloride solution in 20-30 
volumes cold Avater, add a few drops phenolphthalein and a sat- 
urated solution Ba(0H)2 until the purple color just forms. 
Centrifuge, decant oft' the clear liquid, wash with cold water 
and recentrifuge, repeating until there is no pink color formed 
by the addition of phenolphthalein in the wash water. Suspend 
the copper hydroxide in about 100 c. c. cold water and add ap- 
proximately 1 c. c. to the cool flasks, shake vigorously, make up 
to the mark, and allow to warm up to the room temperature. Fil- 
ter through No. 589 blue ribbon filter, pipette oft' 50 c. c. of the 
filtrate and determine the copper complex present as shown be- 
low as a measure of the amino nitrogen. Pipette off 40 c. c. of 
the filtrate for the determination of the polypeptid nitrogen. 

Polypeptid nitrogen: Hydrolize the polypeptids into amino 
acids by adding approximately 5 c. c. concentrated HaSO;^ to 
the 40 c. e. and placing under a steam pressure of 8-10 pounds 
for 10-12 hours. Remove the excess acid with a saturated solu- 
tion Ba(OH)o keeping the solution slightly alkaline to phenol- 
phthalein, filter and wash with carbonated water at least three 
times. Evaporate the filtrate to about 35 or 40 c. c, place in 
100 c. c. measuring flasks, neutralize with N/10 HCl, add 40 c. c. 
buffer solution, 1 c. c. of the copper hydroxide solution in the 
cold water as for the amino determinations and determine the 
copper present in the same manner. 

Copper determination: Place the beakers containing the 50 
c. c. on the hot plate, heat to boiling and neutralize with dilute 
HNO3. Boil down to about one-half and add bromine water 
until a decided bromine color appears, evaporate to about 10-15 
c. c, add 20-30 c. c. pure water and a little more bromine water 
and evaporate down again to 10-15 c. c. Cool, add 2-3 c. c. 
glacial acetic acid, a few crystals potassium iodide, a few drops 
of starch solution and titrate immediately with .001/N sodium 
thio-sulfate until the blue color disappears. Each c. c. of the 
.001/N thio-sulfate solution is the equivalent of 0.000028 gr, 
amino acid nitrogen. 



58 



Preliminary determinations : In addition to the work on the 
soils, an unsuccessful attempt was made to determine tlie amount 
of non-protein and amino acid nitrogen fixed by the ba-ter:a in- 
oculated into the dextrose solution used in the other experiments. 
250 e. c. of the dextrose solution was inoculated with the organ- 
isms indicated in Table 14 and incubated three weeks at room 
temperature. Enough c. p. sodium hydroxide w^as added di- 
rectly to the solution to make 1.5% and the determination car- 
ried out in the above manner. A slight trace was the greatest 
amount found. 

This table shows a decided increase in the soils under field 
conditions over the same soils in the dry state, the greatest in- 
crease taking place during the earlier periods of growth. The 
results of these determinations are grouped in three tables, each 
showing the amount of the different nitrogenous forms found at 
the end of each growing period. 

Discussion of results : A comparison of the results given in 
Tables XVI, XVII and XVIII, shows that there was a definite 
variation of the nitrogenous forms with the length of the time 
of cropping. In almost every case the amount extracted by the 
acid varied Avith the length of time that the soil had been cropped, 
growing smaller and smaller, and the amino and polypeptid 
nitrogen gave similar results. The amount of the^e nitrogenous 

'MB/LlE XIV— lAlMINO' AlCID AND' NON-PiRlOT'EIN" NITKIO'GEN FIXED' BY THE PUlBE 
OULa'UIREiS' IN vSO'I/UTION. 



-id 

E 


Inoculum 


Non-protein N. 


Amino Acid N. 


1 


A. chroococcum CBCIMI- _-_ 


trace 
tracs 




s? 


A. chroococcum CHCIM) 




« 


A. chroococcuin (DfflCIM) - 




4 


No. 26 




5 


No. 26 




fi 


No. 26 




7 


A. chroocoecum C.HCM) and 
A. ehroocoecum CHCIM) and 
A. ehroocoeeum (HCM) and 


No. 26 




8 
9 


No. 20 

No. 28— 


trace 



TAKLE' XV— THE' AMOHINT O'V iDIFFlEKENT NITKlOGENOTJiSi PORlMS IN THE SOIL 
AT THE' ■BElGiINNKNO OE' THE' 'EXIPEIRIIlM'ENTS, ALISO THE SAMIE SOIL PLtJiS 
the: EQUIVALEINT' OE' EH'K TOMS 01ROX.TND' OATS', STKAW OK GlROI]NI> 
OLOVEiR HAY ADIDEB' TO' 'J'll i: s.\.\I l"LE'. DEiTE'BMINATIONS BASED ON THE 
AiMOiUNT' IN 2ft GlR;. 'OiF' 'TH'i': S.\:iIJ''lvl<7 AND' iRIEIS'UlJT'Si ,KXPIRES!SED IN (MG. 
NTTROG'EflSr AND' IN PEK OENT' O'P THE TO'TALi NTTIBOGEN. 



Soil 


OZ8 




c 


1 '^ 

o c 

o.S ti 
Zi ii E 




< « E 


£ 


"o-O M 

ix-n E 


c 


Original . _ _ _ - 


12.95 
IS. 75 

15.40 


1.1243 
1.1666 

1.4424 


8.7 

8.5 

9.3 


2.22T5 
2.339'0' 

2.3475 


17.2 
17.0 

15.2 


O.CS40 
0.1025 

0.1050 


0.7 
0.7 

0.7 


0. 210O 
0.2550 

0.2625 


1.6 


Original + oatS— _ 
Original + red 
clover hay 


1.8 
1.7 



59 



TABLE SVT— (AIMOTHSTTS OF T!H'E iDIFFEIRENT FOKlMiSi OP NffTElOOEJST IN THE 
CRIOOPP'BID ANJy FALLOW Ii:N OiOULAT'EID SOILiS AT TCtlEi END OP THE PIBlST 
PEIBTOID OP GBOWTIH. (BEISUlLrPS EXPIRESBEID IN M'G. MT'KOiGEN FOUND 
AND' IN PEiB CENT OP T'HEI T'O'TIAIj NITfilOGlEIN' CIONTENIT, BASED ON 25 GR. 
SAMIPLE. 



Pots 


5K. 

2 2 M 




1 


2 ti 

C r- C 

0.~ 3 


i 


° 6^3 


c 


Ih 


c 




H S^ 


< t:^ 


c^ 


k, ii3 


Ph 


< ^3 


Oh 


£i.° 


IX 


9— ll-_. 


14.33 


2.0242 


14.1+ 


3.1675 


22.0 


0.1260 


0.8+ 


0.4725 


3.2+ 


10—12— 


15.60 


1.9181 


12.3— 


2.6650 


17.0+ 


0.1505 


0.9+ 


0.2975 


1.9 


13—15— 


20.39 


2.2151 


10.8 + 


2.3690 


11.6+ 


0.1400 


0.7— 


0.4aS0 


2.2 + 


14—16— 


19.81 


1.9S18 


10.0+ 


3.0650 


15.4+ 


0.1085 


0.5+ 


O.2O0O 


1.0+ 


17—19— 


23.03 


2.2939 


9.91+ 


3.5000 


15.2+ 


0.1015 


0.4+ 


0'.2625 


1.1 + 


IS— 20l__ 


23.09 


1.1121 


4.7 + 


2.8500 


a2.3i+ 


0.1680 


0.7 + 


0.2100 


0.9+ 


21— 23-._ 


19.77 


2.3424 


11.8+ 


2.7175 


13.8— 


0.2810 


1.1 + 


0.4200 


2.1 + 


22—24— 


20.19 


0.S648 


4.8+ 


21.7325 


13.5+ 


0.1400 


0.7— 


0.3500 


1.7+ 


25—27 


17.50 


2.4030 


13.7+ 


2.6950 


15.4 


0.1820 


1.0 + 


0.4375 


2.5 


26—28-.. 


22.54 


1.9878 


8.S+ 


2.7425 


12. a+ 


0.2110 


0.9+ 


0.31.50 


1.4— 


29^31... 


20.94 


2. 3848 


11.4— 


2.5150 


12.0+ 


0.1750 


0.8+ 


0.2100 


1.0 


30^-S2... 


118.69 


0.7515 


4.1— 


2.5875 


13.8+ 


0.09SO 


0.5+ 


0.3200 


1.7+ 



TABLE XVn— AM'O'UNTISi OP THE DIPPEBENT PIOIRMISI OP NTTEOGEN IN THE 
PALLO'W AND ORiOiPPED' IXOOULATE© SOIL® AT THE: END OP THE SECOND 
G'RiOiWING PERIOD. RESIULTS EXPRES'^'ED' IN M!G. N. POUND AND' IN PEK 
CENT OP THE TOTAIL N. CONTENT EASED O'N 25 -GEiAM SAiMP'LflB. 



Pots 


„ c . 

2 g M 


1 bi 
'G re 3 


c 


2 M 
g.S§ 






a 


0-T3 <^ 


*-' 




H SS 


< i:3 


Oh 


2^ 2^ 


o. 


< « S 


CU 


£-Z B 


0- 


9-11... 


16.45 


1.6515 


10.0 


1.8675 


11.1+ 


O.O70O 


0.4+ 


0.2800 


1.7+ 


10—12... 


15.44 


lost 





3.41S0 


22.1 + 


0.1110 


0.T+ 


0.3675 


2.4^ 


13-15... 


21.48 


2.1896 


10.2 


2.0700 


9.0+ 


0.1260 


0.6— 


O.160O 


0.7+ 


14—16 


19.32' 


0.7257 


3.V+ 


3.99'2S 


15.5— 


O.140O 


0.7+ 


0.1225 


0.6+ 


17—19 


16.62 


0.9212 


5.5+ 


2.6825 


16.1 + 


0.4750 


2.8+ 


0.1750 


1.1— 


18^20 


15.84 


0.7257 


4.6— 


3.4075 


21.5+ 


O.02O1 


0.1 + 


0.3300 


2.2+ 


21—23 


19.74 


1.7S95 


9.1— 


3.5000 


17.8— 


0.1190 


0.6— 


0.2100 


1.0+ 


22—24 


19.78 


0.7500 


. 3.8— 


3.33OT 


16.8+ 


O.063O 


0.3 + 


0.2800 


1.4 + 


25—27-.. 


17.28 


1.4727 


8.5+ 


2.6370 


15.3— 


0.2660 


1.5+ 


0.2100 


1.2+ 


26—28 


16.32 


0.6030 


3.7— 


2.6000 


15.9+ 


0.0630 


0.4— 


0.29'76 


1.8+ 


29—31... 


20.31 


2.1363 


10.5+ 


2.8330 


13.9+ 


O.091O 


0.4+ 


O.280O 


1.3+ 


30—32 


20. 6® 


1.1000 


5.4 + 


3.4900 


16.9— 


0.0'700 


0.3+ 


0.2275 


1.1 + 



TABLE XVIIT— AMIO'UNTB OP THE DrPPEiBENT POIRMS OP' NITROGEN IN THE 
PALL'OW ANiD CiROiPPED INOCiULA,T'EID' iSiOTLlSi AT THEi EINlD OP THE THIBD 
AND LAST GR'O'WTNIG PEIRiTOD. iREISUODTS IBABED' ON 23 GIEAM S'AIMPLE, 
EXPRESSED IN IMG. N. POUND' u-m-D IN PEiR CENT O'P TOTAL N. CONTENT. 



Pots 


Total N. 

content 

2Sgr. 




c 

u 


1 TJ 

o c 
2^ £ E 


c 


'Ev. 'i 

< « E 


c 


a 

0-T3 5 

c^-s3 


c 
£. 


9^11... 


16.93 


1.2833 


7.5+ 


2.1475 


12.7— 


0.0420 


0.2+ 


0.2275 


1.3+ 


10—12... 


15.65 


0.5773 


3.7 — 


3.2850 


20.9+ 


0.0490 


0.5+ 


O.210O 


1.3 + 


13—15... 


20.23 


0.7485 


S.7— 


2.3675 


11.2+ 


O.042O 


0.2+ 


0.2450 


1.2+ 


14—16... 


18.19 


0.6306 


3.4+ 


3.3005 


18.1 + 


O.091O 


0.4t 


O.2100 


1.1 + 


17-^19... 


16.90 


1.1773 


e.9+ 


2.7025 


15.9+ 


0.0210 


0.1 + 


0.2625 


1.6— 


18—20..- 


14.94 


0.5560 


3.7+ 


3.6823 


24.7— 


0.06.30 


0.4+ 


O.140O 


0.9+ 


21—23 


22.03 


1.7727 


8.1— 


2.8560 


VZ.9+ 


0.0420 


0.2'— 


0.2800 


1.3— 


2r2-24-.. 


18.21 


0.7000' 


3.8+ 


3. 03 GO 


16.5+ 


0.3240 


1.8— 


0.1750 


0.9+ 


25—27 


a6.7l 


1.506O 


9.0+ 


2.6925 


16.1 + 


0.0350' 


0.2+ 


0.5950 


3.6— 


26^28 


15.66 


0.7212 


4.C + 


3.6350 


23.2+ 


0.0420 


0.31— 


0.3150 


2.0+ 


29-31— 


20.36 


2.2060 


10.8+ 


2.740O 


13.4+ 


O.056O 


0.4 + 


0.7075 


3.4+ 


3D--32 


18.091 


0.5939 


3.3— 


3.4500 


11.3— 


0.0770 


0.4+ 


0.2800 


1.5 + 



60 

compounds became smaller, as decomposition of the oro:anic mat- 
ter proceeded, at a sligiitly faster rate than the total nitrogen 
content of the soil became depleted. The non-protein nitrog'eu 
also varied considerably, altho' not in the marked degree shown 
by the other forms. Neither the oats straw or the red clover hay, 
added as manures to the pots, showed any effect on the forms of 
nitrogen determined, further than the small amount shown in the 
preliminary determinations. If there was a difference in the 
soil under field conditions it evidently was too small to he 
measured by these methods. It is entirely possible that the 
amounts of these complex nitrogenous compounds are rapidly 
changing into other forms and that the per cent they bear to the 
total nitrogen content remains somewhat constant, varying only 
with the amount of organic matter present in the beginning, then 
as decomposition proceeds and the more complex combinations 
are broken up, this percentage relation becomes smaller and 
smaller until it reaches a constant. 

Once decomposition had begun in the soil there was absolutely 
no tendency for the more complex nitrogenous forms to accumu- 
late under conditions approximating those in the field. Instead of 
an accumulation there was a steady reduction. How closely this 
reduction is coupled with the decay of the organic matter and 
what would be the final equilibrium between the total nitrogen 
content and the nitrogenous compounds are questions for further 
study. 

Summary. 

1. The acid extracted, non-protein, amino and polypeptid 
nitrogen changed into other forms with the advance of decompo- 
sition much faster than the total nitrogen contents of the soils 
in question decreased. 

2. Oats straw and clover hay added as manures at the rate 
of five tons per acre had little effect in influencing this change. 

3. The amounts of non-protein and amino acid nitrogen fixed 
by bacterial cultures in solution were negligible. 

4. Bacterial inoculation had apparently no effect on the 
amounts of non-protein, amino or polypeptid nitrogen in the soil. 

5. There was no tendency for the above forms of nitrogen to 
accumulate in the soil under conditions approximating those in 
the field. 

Acknowledgments: I wish to express my thanks to Dr. P. E. 
Brown for his help and suggestions thruout this work and to Dr. 
H. S. Potter for his suggestions in the determinations of the com- 
plex forms of nitrogen. 



APPENDIX TABLE 1. 



===== 


== 


= ^ 




Found in Tot Soils, Cniculated 


1 


== 


—^ 












on B 


osia of 4491 vj. 'n Fallow and 4536 gr. 


1 








S = ° 






1 
1 


liind of Crop 
Grown 




in 


cropred.— 


P..t 




Ji 




m 

izi 


2 


-"lias 

„ % c 0.^ 


n-c 


Inoculum Used and 
Pot No. 


Determinations 


1 

fi 


2 

[2 


11 


1 


Q 


> 


11.1 

ill 



i,chrooc. (HCM) 


F 

P 





3.0309 
2.7668 
4.2744 
4.H171 


3.3630 
3.2079 
4.3373 
4.2547 


3.3499 
2.9SS3 
4.305S 
4.1809 


"oroiio" 


3.54SD 
2.9747 
4.3056 
4.1073 


U.835«" 
7.1150" 


6.0123" 
o.irai" 


3.S499 
2.9870 
4.3058 
4-2684 


3.3833 
2.9S70 
4-3488 
4.2684 


3.6232 
3.7309 
3.8963 
4.0046 












Clover 

dover 




e'. {.'.'.'.'— — 




0.2638 


A, c/l">-toi.i""' 


F 

F 





■'.SCOl 
3.4450 
4.6663 
■1.4770 


3.6S20 
3.0310 
4.57S)S 
4.50S7 


3.2210 
S.53S0 
4.5726 
4.492S 


""o'oisis" 

0.0136 


3.2210 
3.6244 
4.5726 
4.1702 


"i.noo"' 
""eSoo"" 


1.0564" 

l^roS" 


3.2210 
3.5808 
4.5726 
4.5787 


3.2532 
3.5S0S 
4.6183 

4.57S7 


3.6232 
3.7309 
3.8963 
4.0046 




11 

ID-IZ 

13-lS 

14-lC 


0at3 

Clover 

Clover 


0.7220 
0.5741 


4. iictjcwckii 

[7-19— 

IS-M- - - 

21-2S 


F 

F 
C 

F 


P 

c 

F 


Oats _ 

Oats 

UKiVcr 

Clover 

Oats ,-- 

Oats 

CiOvcr -- 

Clover -.- 

Oats 


5.1230 
5.1392 
4.5102 
4.b8Sl 

S.OOOl 
5.17.55 
2. iOol 
4.2S05 

3.4tSV 


5.2173 
6.3343 
4.3CS7 
4.6722 

3.8973 
6.0-185 
2.44V4 
4.1912 

3.4SS7 


5.1701 
5.28617 
4.4394 
4.E8C1 

3.9267 
5.1120 
2.4572 
4.23SS 

3.4SSr 


"i'.mii' 
"o.oise" 

"aoiss" 
"oroise"' 


5.1701 
5.2731 
4.4394 
4-5666 

3.0267 
5.0984 
2.45r2 
4-2252 

3.48S7 


""o^Sso"" 
"7?oi6o"" 

"i'.'dim" 
S.2050"' 


"6.6093" 
o.oSs" 

0.0810 
"6.1205 


5.1701 
5.2S24 
4.4394 
4.6613 

3.9267 
6.1791 
2.4S72 
4.3467 


5.2218 

6.as24 

4.4S27 
4.6613 

3.9659 
5.1794 
2.4817 
4.3457 


3.6232 
3.7309 
3.8963 
4.00-16 

3.ia32 
3.731-.' 
3.'S«i 
4-l«16 


1.5086 
1.5515 
0.5SC4 


25-27- 

20-28 — - 

20-31 — - 


0.3427 
1.44S5 


Az. seD 

,\3-»5 







Oats 








0.01-36 


3.S034 


0.4900 












•n 19' 


1' 


Glover 










4.4823 














SS-40 





Olovcr 








6.0136 


4.55S6 


■t.VXili 


6.1247 


4-6833 


4.0SS3 


4.0046 


0.6/87 




F 



Oats --- 

Onts -. 


3.5S30 
3.556-2 


3.70S7 
3.5S79 


3.6458 
3.5/20 


"oToiseT" 


3.6458 
.1.5584 


i'.im' 




3.6158 


3.6^22 


3.0232 










F 


Clover _ - 











4.5023 














l6-(8 — 





Clover 






4.3114 


0.0136 


4-2973 


9.(H60 


0.1217 


4.4195 


4.41i;5 


4.0046 


0.4149 




F 


Oats 


3.3«30 


3.4573 


3-41(1 




3.4101 








3.4442 











Oats 






3.6323 


0.0136 


3.5187 


2. 1850 




3.5519 


3.5519 


3.7309 






F 


Clover - 






4-4001 




4.40.11 














51-56 


C 


aovcr - 


4.272SI 


4.S0OO 


4.3114 


0.0136 


4.2978 


6.7850 


0.08S8 


4.3866 


4,3Mi6 


4.0040 


0.3t20 








3.6M4 


3.59S7 












3.6065 
3.5323 






0.0:33 





Oats 


3.4'.i27 


3.5085 


3.5006 


0.01S6 


S.4S10 


3.8650 




3. 5323 


3.730') 




F 


Clover ..- 


4.^.310 


4.4C44 


4.4630 


._. 


4.4630 






4.4630 


4..i076 


3.8903 


V.6113 


62-(H 

Wis".d cultures 

00-07 





Clover 


4.2230 


4.3817 


4.3023 


O-0136 


4.2fS7 


7.1400 


0.107S 


4.3905 


4.3905 


4.O0W 


H.3919 































Oats 


3.5563 


3.6514 


3.603S 


0.0136 


3.5902 


1-0450 




3.1:021 


3.6021 


3.7109 






F 


Clover 






4.3087 




4.3687 






4.3ia7 


4.4123 


3.SJ63 


0.516(1 




C 


Clover .— 


4.4135 


4.5105 


4.4720 


0-0136 


4.4684 


9-530O 


i.l210 


4.57114 


4.5794 


4.0046 


0.5748 


Check 
























Average 






P 


Nothing _.- — 


3.6369 


5.5515 


3.5912 




3.594'. 






3.5942 


3.6301 


3.4816 




75-76 — 





Nothing 


3.5562 


3-4609 


3.5CS5 


0-0136 


3.4949 


5.5650 


0.0740 


3.5689 


3.56S9 


3.5S93 






V 


Nothing 


3.S002 


3.3002 


3.3002 




3.3002 






3.3002 


3.3332 






JS-90 - — 





Nothing 


3.4609 


3.6197 


3.5103 


0.0136 


3.6267 


C.7700 


0.0830 


3.6097 


3.6097 








APPENDIX TABLE II. 



Actual Grams N. Found in Pot Soils, Calculated 

on Basis 41J6 gr. in Fallow, 4101 gr. in 

Cropi ed. — Pots 

Determination 







, 




c 












S.5 


SZ°^ 


Hi 


!|i 




ttoS.g 


g-ss 


oP°S6 


•3.3-S 






o 


-^1 


2.7340 


3.0128 


3.3370 


2.8S64 


3.0910 


3.1866 


3.3205 


3.6592 


3.6107 


3.3397 


3.6102 


3.4603 


2.6867 


2.9607 


3.3370 


2.5610 


2.7947 


3.1866 


3.5242 


3.8S36 


3.6107 


3.2020 


3.4901 


3.4003 


2.7165 


2.9924 


3.3370 


2.6263 


2.86-26 


3.1866 


3.3343 


3.6743 


3.6107 


3.2897 


3.5857 


3.4003 


2.8235 


3.1115 


3.3370 


2.7306 


2.9763 


3.1866 


3.3205 


3.6591 


3.6107 


3.463(5 


3.6322 


3.4603 


2.7517 


3.0356 


3.3370 


2.9120 


3.1730 


3.1866 


3.4921 


3.8492 


3.6107 


3.0901 


3.S747 


3.4603 


2.7015 


2.9770 


3.3370 


3.0069 


3.3J"9 


3.1806 


3.4487 


3.S0O4 


3.6107 


3.5425 


3-8613 


3.4603 


2.70S3 


2.9845 


3.3370 


2.9083 


3.17(0 


3.1866 


3.3977 


3.7442 


3.0107 


3.2959 


3.69-25 


3.4603 


2.6146 


2.S812 


.1.3370 


2.8681 


3.1-202 


S.lwd 


3.3998 


3.7465 


3.6107 


3.2031 


3.4913 


3.4003 


2.5210 


2.7781 


3.3370 


2.7221 


2.9310 


3.186'j 


3.2696 


3.0031 


3.01/7 


3.4091 


3.7159 


3.4 .(3 
Aver!i:--e 


2.8235 


3.1115 






3.0537 




2.9768 


3.2793 


t 


2.7857 


3.0364 


II 



Onts ... 
C?lovt.r . 



Oats .. 
Oats ... 
Clover . 
Olovcr . 



Oats . . . 
Clover ... 
Clover . . 

Oats ... . 
Oats .... 
Clover .- 
Clover .. 

Oats ... 
Oats .... 
Clover .- 
Clover _. 

Oats .... 
Oats .... 
Clover .. 
Clover .. 

Oata .... 
Onts .... 
caover .. 
Clover .. 



Nothing . 
Nothing 



2.7517 
2.85.37 
3.6443 



2.7227 
2.SCS3 
3.3977 
3.3051 



2-8001 
2.75IS 
2.9696 
2.fl»5 



2.7900 
3.2557 
3.2177 



2.7083 
2.5910 
3.4318 
3.3(ftl 



2.7577 
2.!I-2S6 
3.4400 
3.04.<10 



2.6790 
3.2096 
3.3183 

2.8S79 
2.7618 
2.9S'20 



2.7340 
2.S21S 
3.3-205 
3.3123 

2.6807 
2.5479 
3.5242 
3.1813 



2.6134 
3.3343 
3.2681 

2.8235 
2.0936 
3.3-205 
3.4143 

2.7.547 
2.S9I2 
3.4921 
8.0618 

2.7015 
3.0503 
3.44S7 
3.50S9 



2.6146 
2.8-246 
3.3908 
3.1740 



2.8235 
2.7518 
2.9758 
2.T7S6 



0.0136 
0.0130 



0.0136 
0.0136 



O.OISO 
6.0136 



0.0136 
'6.6l36 



0.0136 
6.0130 

"o.6i36 
"616136" 

"6.61S6 
0.0130 

6.0136 
0.0136 



3.2445 
2.8235 



2.7547 
2.8776 
8.4921 
3.0512 



2.8748 
3.3977 
3.2531 



2.8235 
2.7382 
2.9758 
2.7600 



"'l?4995 " 


' 016282'" 


"2?i746" 


"6"64i6"" 


"i.7S7"" 


"T6297" 


"ir6253" 


""6!m43" 


1.8895 


0.0205 


i?S346" 


"016452" 


"2"ol7s'" 


6.6566" 


"s^nss"" 


""6^629"" 


"2?2545" 


6?6S4"' 


2.2800 


0.0149 


"i"6s66'" 


"'6S362" 


"I'sffls" 


"6^6472" 


"'i'so-Is'" 


"6'6336"" 


2.6103 


0.0428 


""s^oior" 


li.mi 


""i^iooo" 


6^0127 


2.2315 " 


0.0422 


1.6933 


0.0376 


'T.m&' 


""6;6ie9" 


"ilm' 


'"o"657"" 



0.1663 
0.1897 
0.4010 



IIJ.O4B0 -t- clover N. -|- 3.4603 



APPENDIX TABLE III. 









Az. 27D 

41 

42 

43 



64 -. 

05 



Ai-iual Grams N. Found in Pot Soils, Calculated 

on Basis of 3741 er. in Fallow and 3786 

gr. in Cropped Pots 



Oats ... 
Oats -. 
Oats ... 
Clover . 
Clover - 
Clover . 
Clover . 



Onts 

Oats 

Oats 

Clover .. 
Olover _- 
Clover .. 
Olover — . 

Oats . , 

Oats 

Oats 

Oats 

Olov(!r ... 
Clover ... 
Clover ... 
Olover ... 

Onts 

Oats 

Oats 

Oats 

Clover ... 

Olover 11' 
Clover ... 

Oats 

Oats 

Oats 

Oats 

Clover ... 
Olover ... 
Olover ._ 
Clover .. 

Oats 

Oats 

Oati 

Oats 

Olover .. 
Clover .. 
Clover _- 
Olover .. 

Oats ._- 

Oats 

Oats 

Oats 

Clover .. 
Olover .- 
Cnover .. 
(/lover .- 

Oata 

Oats 

Oats .... 

Oats 

Clover .. 
Olover .- 
Clover .. 
(plover .- 

Oats 

Oats 

Oata -. 
Oats „-. 
(Jlovor .^. 
Clover .. 
Olover _. 
Olover .. 

Nothing 
Nothing 
Nothing 
Nothing 
Nothing 
Nothing 
Nothing 
Nothing 



2.S30O 
2.8131 
2.7619 
2.1864 



2.3321 
2.3038 
2.3586 
2.9059 
2.S751 
3.1808 
2.5971 

2.5018 



2.81S9 
3.0499 
2.8773 



2.6574 
2.1074 
2.4216 



2.9583 
3.2ni 
2.8405 



2.5656 
2.32-27 
2. 4478 
3.0070 
3.5203 
2.7129 
3.0761 
2.7694 

2.5731 
2.3983 
2.4478 
2.3719 
3.0-237 
2.7429 



2.4740 
2.5044 
2.3954 
2.3050 
2.9714 
2.7969 
2.0027 



2.4478 
2.4663 
2.4871 
2.4640 
2.8405 
2.9548 
3.063O 



2.4478 
2.5713 
2.1991 



2.3321 
2.4497 
2.6971 
3.0237 
2.9152 
2.^71 



2.7959 
2.8274 
3. 0666 



2.1074 
2.3954 
2.4616 



3.-2852 
2.8274 
2.8688 

2.7189 
2.36-52 
2.3103 
2.3785 
2.89-28 
2.8489 
3.1646 
2.7501 

2.57S7 
2.2913 
3.4805 



2.5131 
2.3917 
2.4478 
2.3491 
3.0172 
2.7429 

s.oni 

2.7363 

2.4009 
2.4848 
2.3823 



2.4478 
2.4583 
2.4871 
2.4917 
2.8143 
3.0181 
3.056-1 



2.4451 
2.7230 
2.7893 
2.S067 



2.3160 
2.4094 
2.6037 
3.0173 
2.8877 
2.8208 



2.3496 
2.4381 
2.3234 



2.3210 
2.0944 
2.7700 
2.6375 
2.1071 
2.4085 
2.4911 
2.4901 



0.0136 
0.6136 
0.0136 

0.0136 

"o'oiso 

0.0130 
"6?6i3«" 

"6?6i36' 



0.0136 

"o'orn" 



0.0136 
0.6136 
"6^136" 



0.0136 

oToise" 



0.0136 



0.0136 

6.6136 
'6"6i3o' 



O.0136 

"6?6ii6' 
"616136" 
0.6136 

"6^6136" 

"6'oi36" 

"6'6i36" 
0.6136 



2.3507 
2.7S12 
2.17t« 
2.9-255 
3.2710 
2.8274 
2.8562 



2.57S7 
2.2807 
2.4S(B 
2.0601 



2.6431 
2.3761 
2.4478 
2.3351 
3-0172 
2.7293 
3.0711 
2.7227 



2.7113 
2.0161 
2.8741 

2.4478 
2.4447 
2.4871 
2.4781 
2.S14S 
3.0015 



2.4961 
2.5444 
2.2180 
2.4315 
2.7230 
2.7757 
2.6607 
2.6100 



2.4085 
2.4775 
2.4765 



3.0050 

3. mo" 

2.5500 
2.7790 
3.7675 



3.1550 
"3"iS6" 
3.8030 



1.1575 



3.4015 
2.2166' 
3.9045 



S.7480 

"i'.imi 



3.4770 
2.9195 



0.0715 
0.0904 



O.0S68 

o.oiio 



0.0975 
'6ru68' 



0.1002 



0.0785 
"6.6512' 
0.1296' 

'6'i69i' 

0.1103 

"o'iisj' 



0.1280 

"6^940' 
6'i263' 

o'issT 
6.1293' 



0.0420 

'6ri6i6' 

'6?i3-27' 
0.10S7 



0.1055 
"0.6664" 



4.3356 j 0.15S5 
2.9196 I 0.113.8 



2.7489 
2.4231 
2.3103 
2.4653 
2.!,l)-28 
2.y(;34 
3.1540 
2.863-1 

2.6787 



2.4178 
2.4617 
3.0172 



2.4478 
2.6650 
2.4871 
2.6261 
2.S143 
3.1106 
3.0564 
2.9705 

2.4901 
2.0393 
2.21S0 
2.6508 



2.9047 
3.3165 



3.0817 
2.9781 
2.9091 
2.9-191 
3.0598 
3.4381 
3.7252 
3.3V59 



2.9091 
3.0730 
3.0109 
3.1695 
:'..4137 
3.7431 



3.027S 
3.1751 
2.0911 
3.0758 



2.8502 
3.S-2&1 
2.8502 



1.3860 
J. 7020 



0.16S0 

i.iioo" 



2.8502 


0.2345 


3.3281 




2.8502 


0.11S9 


3.3284 




3.1239 


0.6369 


3.0021 




3.1239 


0.0013 


3.00-21 





2.8602 


0.1348 


3.3281 




2.S0O2 


0.0385 


3.S-2S4 




3.1239 


0.40-23 



3.3284 
2.8602 
3.3-28.1 
3.1239 
3.6U21 
3.1239 
3.C021 



S.82S1 
3.1239 
3-6021 



-'\.v. ck. fallow 2.70S6 -f N. c 

Av. ck. fa.llow 2.7086 + N. c 

jVv. cU. cropped 3.1.SfiS -t- N. 

Av. ck. cropped 3.1868 + N. 



ntent of oat.s ground 0.1410=2.8502 
nlcnt ground clover 0.4163 = 3.1239. 
•ontent ground oats 0.1416=3.3284. 
■ontent ground clover 0.4163=3.0021 



*• 



61 



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62 

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